Thrombin-Binding Antibody Molecules and Uses Thereof

ABSTRACT

This invention relates to isolated antibodies which recognise the exosite 1 epitope of thrombin and selectively inhibit thrombin without promoting bleeding. These antibody molecules may be useful in the treatment and prevention of thrombosis, embolism and other conditions mediated by thrombin.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/206,896 filed on Jul. 11, 2016, which is a divisional of U.S. patentapplication Ser. No. 14/309,403 filed on Jun. 19, 2014, which is acontinuation in part of U.S. patent application Ser. No. 14/363,514,filed on Jun. 6, 2014, which is an application filed under Section 371of International Patent Application No. PCT/GB2012/053140, filed Dec.14, 2012, which claims benefit of priority to GB 1121513.4, filed Dec.14, 2011. The contents of these applications are hereby incorporated byreference in their entireties.

REFERENCE TO A SEQUENCE LISTING

This application includes a Sequence Listing submitted electronically asa text file named Thrombin ST25, created on Oct. 27, 2015 with a size of14,000 bytes. The Sequence Listing is incorporated by reference herein.

FIELD OF THE INVENTION

This invention relates to antibody molecules that inhibit thrombin.

BACKGROUND OF THE INVENTION

Blood coagulation is a key process in the prevention of bleeding fromdamaged blood vessels (haemostasis). However, a blood clot thatobstructs the flow of blood through a vessel (thrombosis) or breaks awayto lodge in a vessel elsewhere in the body (thromboembolism) can be aserious health threat.

A number of anticoagulant therapies are available to treat pathologicalblood coagulation. A common drawback of these therapies is an increasedrisk of bleeding (Mackman (2008) Nature 451(7181): 914-918). Manyanticoagulant agents have a narrow therapeutic window between the dosethat prevents thrombosis and the dose that induces bleeding. This windowis often further restricted by variations in the response in individualpatients.

SUMMARY OF THE INVENTION

The present invention relates to the unexpected finding that antibodymolecules which recognise the exosite 1 epitope of thrombin selectivelyinhibit thrombin without promoting bleeding. These antibody moleculesmay be useful in the treatment and prevention of thrombosis, embolismand other conditions mediated by thrombin.

The invention encompasses the following items:

1. An isolated antibody molecule that specifically binds to the exosite1 region of thrombin.

2. The antibody molecule according to item 1 that inhibits thrombinactivity.

3. The antibody molecule according to item 2 which causes minimalinhibition of haemostasis and/or bleeding.

4. The antibody molecule according to item 2 or item 3 which does notinhibit haemostasis and/or cause bleeding.

5. The antibody molecule according to any one of the preceding itemswherein the antibody molecule comprises an HCDR3 having the amino acidsequence of SEQ ID NO: 5 or the amino acid sequence of SEQ ID NO: 5 withone or more amino acid substitutions, deletions or insertions.

6. The antibody molecule according to item 5 wherein the antibodymolecule comprises an HCDR2 having the amino acid sequence of SEQ ID NO:4 or the amino acid sequence of SEQ ID NO: 4 with one or more amino acidsubstitutions, deletions or insertions.

7. The antibody molecule according to item 5 or item 6 wherein theantibody molecule comprises an HCDR1 having the amino acid sequence ofSEQ ID NO: 3 or the amino acid sequence of SEQ ID NO: 3 with one or moreamino acid substitutions, deletions or insertions.

8. The antibody molecule according to any one of items 1 to 7 whereinthe antibody molecule comprises a VH domain having the amino acidsequence of SEQ ID NO: 2 or the amino acid sequence of SEQ ID NO: 2 withone or more amino acid substitutions, deletions or insertions.

9. The antibody molecule according to any one of items 1 to 8 whereinantibody molecule comprises LCDR1, LCDR2 and LCDR3 having the sequencesof SEQ ID NOs 7, 8 and 9 respectively, or the sequences of SEQ ID NOs 7,8 and 9 respectively, with one or more amino acid substitutions,deletions or insertions.

10. The antibody molecule according to any one of items 1 to 9 whereinthe antibody molecule comprises a VL domain having the amino acidsequence of SEQ ID NO: 6 or the amino acid sequence of SEQ ID NO: 6 withone or more amino acid substitutions, deletions or insertions.

11. The antibody molecule according to any one of items 1 to 10comprising a VH domain comprising a HCDR1, HCDR2 and HCDR3 having thesequences of SEQ ID NOs 3, 4 and 5, respectively, and a VL domaincomprising a LCDR1, LCDR2 and LCDR3 having the sequences of SEQ ID NOs7, 8 and 9, respectively.

12. The antibody molecule according to item 11 comprising a VH domainhaving the amino acid sequence of SEQ ID NO: 2 and a VL domain havingthe amino acid sequence of SEQ ID NO: 6.

13. The antibody molecule according to any one of items 1 to 12comprising one or more substitutions, deletions or insertions whichremove a glycosylation site.

14. The antibody molecule according to item 13 comprising a VL domainhaving the amino acid sequence of SEQ ID NO: 6 wherein the glycosylationsite is mutated out by introducing a substitution at N28 or S30.

15. An antibody molecule which competes with an antibody moleculeaccording to any one of items 5 to 12 for binding to exosite 1.

16. The antibody molecule according to any one of items 1 to 15 which isa whole antibody.

17. The antibody molecule according to item 16 which is an IgA or IgG.

18. The antibody molecule according to any one of items 1 to 15 which isan antibody fragment.

19. A pharmaceutical composition comprising an antibody moleculeaccording to any one of items 1 to 18 and a pharmaceutically acceptableexcipient.

20. An antibody molecule according to any one of items 1 to 18 for usein a method of treatment of the human or animal body.

21. An antibody molecule according to any one of items 1 to 18 for usein a method of treatment of a thrombin-mediated condition.

22. Use of an antibody molecule according to any one of items 1 to 18 inthe manufacture of a medicament for use in treating a thrombin-mediatedcondition.

23. A method of treatment of a thrombin-mediated condition comprisingadministering an antibody molecule according to any one of items 1 to 18to an individual in need thereof.

24. An antibody molecule for use according to item 21, use according toitem 22 or method according to item 23, wherein the thrombin-mediatedcondition is a thrombotic condition.

25. An antibody molecule for use, use or method according to item 24wherein the thrombotic condition is thrombosis or embolism.

26. An antibody molecule for use according to item 21, use according toitem 22 or method according to item 23 wherein the thrombin-mediatedcondition is inflammation, infection, tumour growth, tumour metastasisor dementia.

27. A method for producing an antibody antigen-binding domain for theexosite 1 epitope of thrombin, the method comprising;

-   -   (i) providing, by way of addition, deletion, substitution or        insertion of one or more amino acids in the amino acid sequence        of a parent VH domain comprising HCDR1, HCDR2 and HCDR3, wherein        the parent VH domain HCDR1, HCDR2 and HCDR3 have the amino acid        sequences of SEQ ID NOS: 3, 4 and 5 respectively, a VH domain        which is an amino acid sequence variant of the parent VH domain,    -   (ii) optionally combining the VH domain thus provided with one        or more VL domains to provide one or more VH/VL combinations;        and    -   (iii) testing said VH domain which is an amino acid sequence        variant of the parent VH domain or the VH/VL combination or        combinations to identify an antibody antigen binding domain for        the exosite 1 epitope of thrombin.

28. A method for producing an antibody molecule that specifically bindsto the exosite 1 epitope of thrombin, which method comprises:

-   -   providing starting nucleic acid encoding a VH domain or a        starting repertoire of nucleic acids each encoding a VH domain,        wherein the VH domain or VH domains either comprise a HCDR1,        HCDR2 and/or HCDR3 to be replaced or lack a HCDR1, HCDR2 and/or        HCDR3 encoding region;    -   combining said starting nucleic acid or starting repertoire with        donor nucleic acid or donor nucleic acids encoding or produced        by mutation of the amino acid sequence of an HCDR1, HCDR2,        and/or HCDR3 having the amino acid sequences of SEQ ID NOS: 3, 4        and 5 respectively, such that said donor nucleic acid is or        donor nucleic acids are inserted into the CDR1, CDR2 and/or CDR3        region in the starting nucleic acid or starting repertoire, so        as to provide a product repertoire of nucleic acids encoding VH        domains;    -   expressing the nucleic acids of said product repertoire to        produce product VH domains;    -   optionally combining said product VH domains with one or more VL        domains;    -   selecting an antibody molecule that binds exosite 1 of thrombin,        which antibody molecule comprises a product VH domain and        optionally a VL domain; and    -   recovering said antibody molecule or nucleic acid encoding it.

29. An isolated antibody molecule that specifically binds to the exosite1 region of thrombin comprising an LCDR1 having the amino acid sequenceof SEQ ID NO: 7 with one or more amino acid substitutions, deletions orinsertions and wherein said LCDR1 has an amino acid substitution ofalanine for serine at the residue corresponding to S30 of SEQ ID NO: 6(SEQ ID NO: 15).

30. The antibody molecule according to item 29 that inhibits thrombinactivity.

31. The antibody molecule according to item 30 which causes minimalinhibition of haemostasis and/or bleeding.

32. The antibody molecule according to item 30 which does not inhibithaemostasis and/or cause bleeding.

33. The antibody molecule according to item 29 wherein the antibodymolecule further comprises an HCDR3 having the amino acid sequence ofSEQ ID NO: 5 or the amino acid sequence of SEQ ID NO: 5 with one or moreamino acid substitutions, deletions or insertions.

34. The antibody molecule according to item 29 wherein the antibodymolecule further comprises an HCDR2 having the amino acid sequence ofSEQ ID NO: 4 or the amino acid sequence of SEQ ID NO: 4 with one or moreamino acid substitutions, deletions or insertions.

35. The antibody molecule according to item 29 wherein the antibodymolecule further comprises an HCDR1 having the amino acid sequence ofSEQ ID NO: 3 or the amino acid sequence of SEQ ID NO: 3 with one or moreamino acid substitutions, deletions or insertions.

36. The antibody molecule according to item 29 wherein the antibodymolecule further comprises a VH domain having the amino acid sequence ofSEQ ID NO: 2 or the amino acid sequence of SEQ ID NO: 2 with one or moreamino acid substitutions, deletions or insertions.

37. The antibody molecule according to item 29 wherein the antibodymolecule further comprises an LCDR2 and LCDR3 having the sequences ofSEQ ID NOs 8 and 9 respectively, or the sequences of SEQ ID NOs 8 and 9respectively, with one or more amino acid substitutions, deletions orinsertions.

38. The antibody molecule according to item 29 wherein the antibodymolecule comprises the amino acid sequence of SEQ ID NO: 6 with an aminoacid substitution of S30A, and optionally one or more additional aminoacid substitutions, deletions or insertions.

39. The antibody molecule according to item 29 comprising a VH domaincomprising an HCDR1, HCDR2 and HCDR3 having the sequences of SEQ ID NOs3, 4 and 5, respectively, and a VL domain comprising an LCDR2 and LCDR3having the sequences of SEQ ID NOs 8 and 9, respectively.

40. The antibody molecule according to item 39 comprising a VH domainhaving the amino acid sequence of SEQ ID NO: 2 and a VL domain havingthe amino acid sequence of SEQ ID NO: 6 with an amino acid substitutionof S30A (SEQ ID NO: 14).

41. The antibody molecule according to item 29 which is a wholeantibody.

42. The antibody molecule according to item 41 which is an IgA or IgG.

43. The antibody molecule according to item 29 which is an antibodyfragment.

44. A pharmaceutical composition comprising an antibody moleculeaccording to item 29 and a pharmaceutically acceptable excipient.

45. A method of treatment of a thrombin-mediated condition comprisingadministering an antibody molecule according to item 29 to an individualin need thereof.

46. The method of treatment of item 45 wherein the thrombin-mediatedcondition is a thrombotic condition.

47. The method of treatment of item 45 wherein the thrombotic-mediatedcondition is thrombosis or embolism.

48. The method of treatment of item 45 wherein the thrombotic-mediatedcondition is inflammation, infection, tumour growth, tumour metastasisor dementia.

49. A method of treatment of a thrombin-mediated condition comprisingadministering a pharmaceutical composition according to item 44 to anindividual in need thereof.

50. The method of treatment of item 49 wherein the thrombin-mediatedcondition is a thrombotic condition.

51. The method of treatment of item 49 wherein the thrombotic-mediatedcondition is thrombosis or embolism.

52. The method of treatment of item 49 wherein the thrombotic-mediatedcondition is inflammation, infection, tumour growth, tumour metastasisor dementia.

53. A method for producing an antibody antigen-binding domain for theexosite 1 epitope of thrombin, the method comprising;

-   -   (i) providing, by way of addition, deletion, substitution or        insertion of one or more amino acids in the amino acid sequence        of a parent VH domain comprising HCDR1, HCDR2 and HCDR3,    -   wherein the parent VH domain HCDR1, HCDR2 and HCDR3 have the        amino acid sequences of SEQ ID NOS: 3, 4 and 5 respectively, a        VH domain which is an amino acid sequence variant of the parent        VH domain,    -   (ii) combining the VH domain thus provided with a VL domain        having an amino acid substitution of alanine for serine at the        residue corresponding to S30 of SEQ ID NO: 6 (SEQ ID NO: 14) to        provide one or more VH/VL combinations; and    -   (iii) testing the VH/VL combination or combinations to identify        an antibody antigen binding domain for the exosite 1 epitope of        thrombin.

54. A method for producing an antibody molecule that specifically bindsto the exosite 1 epitope of thrombin, which method comprises:

-   -   providing starting nucleic acid encoding a VH domain or a        starting repertoire of nucleic acids each encoding a VH domain,        wherein the VH domain or VH domains either comprise a HCDR1,        HCDR2 and/or HCDR3 to be replaced or lack a HCDR1, HCDR2 and/or        HCDR3 encoding region;    -   combining said starting nucleic acid or starting repertoire with        donor nucleic acid or donor nucleic acids encoding or produced        by mutation of the amino acid sequence of an HCDR1, HCDR2,        and/or HCDR3 having the amino acid sequences of SEQ ID NOS: 3, 4        and 5 respectively, such that said donor nucleic acid is or        donor nucleic acids are inserted into the CDR1, CDR2 and/or CDR3        region in the starting nucleic acid or starting repertoire, so        as to provide a product repertoire of nucleic acids encoding VH        domains;    -   expressing the nucleic acids of said product repertoire to        produce product VH domains;    -   combining said product VH domains with a VL domain having an        amino acid substitution of alanine for serine at the residue        corresponding to S30 of SEQ ID NO: 6 (SEQ ID NO: 14);    -   selecting an antibody molecule that binds exosite 1 of thrombin,        which antibody molecule comprises a product VH domain and a VL        domain having an amino acid substitution of alanine for serine        at the residue corresponding to S30 of SEQ ID NO: 6 (SEQ ID NO:        14); and    -   recovering said antibody molecule or nucleic acid encoding it.

The present invention further provides recombinant expression vectorsengineered to express the antibodies of the present invention asdescribed above, including for example those antibodies having the S30Asubstitution. Such expression vectors and their uses are well known tothose of skill in the art. In an embodiment of the invention theexpression vector may be one designed for expression of a protein ofinterest, such as an antibody molecule, or fragment thereof, inprokaryotic cells such as bacteria or eukaryotic cells such as mammaliancells. In a specific embodiment of the invention the expression vectormay provide for protein expression in CHO cells.

The invention encompasses the additional following items:

55. A recombinant expression vector encoding for an isolated antibodymolecule that specifically binds to the exosite 1 region of thrombin.

56. The recombinant expression vector according to item 55 comprising anLCDR1 having the amino acid sequence of SEQ ID NO: 7 with one or moreamino acid substitutions, deletions or insertions and wherein said LCDR1has an amino acid substitution of alanine for serine at the residuecorresponding to S30 of SEQ ID NO: 6 (SEQ ID NO: 15).

57. The recombinant expression vector according to item 56 wherein theantibody molecule further comprises an HCDR3 having the amino acidsequence of SEQ ID NO: 5 or the amino acid sequence of SEQ ID NO: 5 withone or more amino acid substitutions, deletions or insertions.

58. The recombinant expression vector according to item 56 wherein theantibody molecule further comprises an HCDR2 having the amino acidsequence of SEQ ID NO: 4 or the amino acid sequence of SEQ ID NO: 4 withone or more amino acid substitutions, deletions or insertions.

59. The recombinant expression vector according to item 56 wherein theantibody molecule further comprises an HCDR1 having the amino acidsequence of SEQ ID NO: 3 or the amino acid sequence of SEQ ID NO: 3 withone or more amino acid substitutions, deletions or insertions.

60. The recombinant expression vector according to item 56 wherein theantibody molecule further comprises a VH domain having the amino acidsequence of SEQ ID NO: 2 or the amino acid sequence of SEQ ID NO: 2 withone or more amino acid substitutions, deletions or insertions.

61. The recombinant expression vector according to item 56 wherein theantibody molecule further comprises an LCDR2 and LCDR3 having thesequences of SEQ ID NOs 8 and 9 respectively, or the sequences of SEQ IDNOs 8 and 9 respectively, with one or more amino acid substitutions,deletions or insertions.

62. The recombinant expression vector according to item 56 wherein theantibody molecule comprises the amino acid sequence of SEQ ID NO: 6 withan amino acid substitution of S30A, and optionally one or moreadditional amino acid substitutions, deletions or insertions.

63. The recombinant expression vector according to item 56 comprising aVH domain comprising an HCDR1, HCDR2 and HCDR3 having the sequences ofSEQ ID NOs 3, 4 and 5, respectively, and a VL domain comprising an LCDR2and LCDR3 having the sequences of SEQ ID NOs 7 and 8, respectively.

64. The recombinant expression vector according to item 63 comprising aVH domain having the amino acid sequence of SEQ ID NO: 2 and a VL domainhaving the amino acid sequence of SEQ ID NO: 6 with an amino acidsubstitution of S30A (SEQ ID NO: 14).

The present invention is also directed to recombinant cells engineeredto express the antibodies of the present invention as described above,including for example those antibodies having the S30A substitution. Inan embodiment of the invention, such recombinant cells may compriserecombinant expression vectors that provide for the expression of theantibody molecules of the present invention in such cells. Recombinantcells may be prokaryotic cells such as bacteria, as well as eukaryoticcells such as mammalian cells. In a specific embodiment of theinvention, the recombinant cells may be CHO cells such as thosedescribed in the working examples of the specification.

The invention encompasses the additional following items:

65. A recombinant cell expressing an antibody molecule that specificallybinds to the exosite 1 region of thrombin.

66. The recombinant cell according to item 65 expressing an antibodycomprising an LCDR1 having the amino acid sequence of SEQ ID NO: 7 withone or more amino acid substitutions, deletions or insertions andwherein said LCDR1 has an amino acid substitution of alanine for serineat the residue corresponding to S30 of SEQ ID NO: 6 (SEQ ID NO: 15).

67. The recombinant cell according to item 66 wherein the antibodymolecule further comprises an HCDR3 having the amino acid sequence ofSEQ ID NO: 5 or the amino acid sequence of SEQ ID NO: 5 with one or moreamino acid substitutions, deletions or insertions.

68. The recombinant cell according to item 66 wherein the antibodymolecule further comprises an HCDR2 having the amino acid sequence ofSEQ ID NO: 4 or the amino acid sequence of SEQ ID NO: 4 with one or moreamino acid substitutions, deletions or insertions.

69. The recombinant cell according to item 66 wherein the antibodymolecule further comprises an HCDR1 having the amino acid sequence ofSEQ ID NO: 3 or the amino acid sequence of SEQ ID NO: 3 with one or moreamino acid substitutions, deletions or insertions.

70. The recombinant cell according to item 66 wherein the antibodymolecule further comprises a VH domain having the amino acid sequence ofSEQ ID NO: 2 or the amino acid sequence of SEQ ID NO: 2 with one or moreamino acid substitutions, deletions or insertions.

71. The recombinant cell according to item 66 wherein the antibodymolecule further comprises an LCDR2 and LCDR3 having the sequences ofSEQ ID NOs 8 and 9 respectively, or the sequences of SEQ ID NOs 8 and 9respectively, with one or more amino acid substitutions, deletions orinsertions.

72. The recombinant cell according to item 66 wherein the antibodymolecule comprises the amino acid sequence of SEQ ID NO: 6 with an aminoacid substitution of S30A, and optionally one or more additional aminoacid substitutions, deletions or insertions.

73. The recombinant cell according to item 66 comprising a VH domaincomprising an HCDR1, HCDR2 and HCDR3 having the sequences of SEQ ID NOs3, 4 and 5, respectively, and a VL domain comprising an LCDR2 and LCDR3having the sequences of SEQ ID NOs 8 and 9, respectively.

74. The recombinant cell according to item 73 comprising a VH domainhaving the amino acid sequence of SEQ ID NO: 2 and a VL domain havingthe amino acid sequence of SEQ ID NO: 6 with an amino acid substitutionof S30A (SEQ ID NO: 14).

75. A recombinant cell comprising the expression vector according toitems 55-64.

DETAILED DESCRIPTION OF THE INVENTION

An aspect of the invention provides an isolated antibody molecule thatspecifically binds to exosite 1 of thrombin.

Isolated anti-exosite 1 antibody molecules may inhibit thrombin in vivowithout promoting or substantially promoting bleeding or haemorrhage,i.e. the antibody molecules do not inhibit or substantially inhibitnormal physiological responses to vascular injury (i.e. haemostasis).For example, haemostasis may not be inhibited or may be minimallyinhibited by the antibody molecules (i.e. inhibited to an insignificantextent which does not affect the well-being of patient or requirefurther intervention). Bleeding may not be increased or may be minimallyincreased by the antibody molecules.

Exosite 1 (also known as ‘anion binding exosite 1’ and the ‘fibrinogenrecognition exosite’) is a well-characterised secondary binding site onthe thrombin molecule (see for example James A. Huntington, 2008,Structural Insights into the Life History of Thrombin, in RecentAdvances in Thrombosis and Hemostasis 2008, editors; K. Tanaka and E. W.Davie, Springer Japan KK, Tokyo, pp. 80-106). Exosite 1 is formed inmature thrombin but is not formed in prothrombin (see for exampleAnderson et al (2000) JBC 2775 16428-16434).

Exosite 1 is involved in recognising thrombin substrates, such asfibrinogen, but is remote from the catalytic active site. Variousthrombin binding factors bind to exosite 1, including the anticoagulantdodecapeptide hirugen (Naski et al 1990 JBC 265 13484-13489), factor V,factor VIII, thrombomodulin (cofactor for protein C and TAFIactivation), fibrinogen, PAR1 and fibrin (the co-factor for factor XIIIactivation).

An anti-exosite 1 antibody may bind to exosite 1 of mature humanthrombin. The sequence of human preprothrombin is set out in SEQ IDNO: 1. Human prothrombin has the sequence of residues 44 to 622 of SEQID NO: 1. Mature human thrombin has the sequence of residues 314-363(light chain) and residues 364 to 622 (heavy chain).

In some embodiments, an anti-exosite 1 antibody may also bind to exosite1 of mature thrombin from other species. Thrombin sequences from otherspecies are known in the art and available on public databases such asGenbank. The corresponding residues in thrombin sequences from otherspecies may be easily identified using sequence alignment tools.

The numbering scheme for thrombin residues set out herein isconventional in the art and is based on the chymotrypsin template (BodeW et al EMBO J. 1989 November; 8(11):3467-75). Thrombin has insertionloops relative to chymotrypsin that are lettered sequentially usinglower case letters.

Exosite 1 of mature human thrombin is underlined in SEQ ID NO: 1 and mayinclude the following residues: M32, F34, R35, K36, S36a, P37, Q38, E39,L40, L65, R67, 572, R73, T74, R75, Y76, R77a, N78, E80, K81, 182, 583,M84, K109, K110, K149e, G150, Q151, 5153 and V154. In some embodiments,other thrombin residues which are located close to (i.e. within 0.5 nmor within 1 nm) of any one of these residues may also be considered tobe part of exosite 1.

An anti-exosite 1 antibody may bind to an epitope which comprises 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or morethan 20 residues of exosite 1. Preferably, an anti-exosite 1 antibodybinds to an epitope which consists entirely of exosite 1 residues.

For example, an anti-exosite 1 antibody may bind to an epitope whichcomprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or all 16residues selected from the group consisting of M32, F34, S36a, P37, Q38,E39, L40, L65, R67, R73, T74, R75, Y76, R77a, 182 and Q151 of humanthrombin or the equivalent residues in thrombin from another species. Insome preferred embodiments, the epitope may comprise the thrombinresidues Q38, R73, T74, Y76 and R77a and optionally one or moreadditional residues.

Anti-exosite 1 antibody molecules as described herein are specific forthrombin exosite 1 and bind to this epitope with high affinity relativeto other epitopes, for example epitopes from mammalian proteins otherthan mature thrombin. For example, an anti-exosite 1 antibody moleculemay display a binding affinity for thrombin exosite 1 which is at least500 fold, at least 1000 fold or at least 2000 fold greater than otherepitopes.

Preferably, an antibody molecule as described herein which is specificfor exosite 1 may bind to mature thrombin but display no binding orsubstantially no binding to prothrombin.

Without being bound by any theory, anti-exosite 1 antibodies may beunable to access thrombin within the core of a haemostatic clot, and aretherefore unable to affect haemostasis by interrupting normal thrombinfunction at sites of vascular injury. However, because the anti-exosite1 antibodies still bind to thrombin on the surface of the clot and inthe outer shell of the clot, thrombosis is prevented, i.e.non-haemostatic clot extension is prevented.

An anti-exosite 1 antibody molecule may have a dissociation constant forexosite 1 of less than 50 nM, less than 40 nM, less than 30 nM, lessthan 20 nM, less than 10 nM, or less than 1 nM. For example, an antibodymolecule may have an affinity for exosite 1 of 0.1 to 50 nM, e.g. 0.5 to10 nM. A suitable anti-exosite 1 antibody molecule may, for example,have an affinity for thrombin exosite 1 of about 1 nM.

Binding kinetics and affinity (expressed as the equilibrium dissociationconstant, K_(d)) of the anti-exosite 1 antibody molecules may bedetermined using standard techniques, such as surface plasmon resonancee.g. using BIAcore analysis.

An anti-exosite 1 antibody molecule as described herein may be animmunoglobulin or fragment thereof, and may be natural or partly orwholly synthetically produced, for example a recombinant molecule.

Anti-exosite 1 antibody molecules may include any polypeptide or proteincomprising an antibody antigen-binding site, including Fab, Fab₂, Fab₃,diabodies, triabodies, tetrabodies, minibodies and single-domainantibodies, including nanobodies, as well as whole antibodies of anyisotype or sub-class. Antibody molecules and methods for theirconstruction and use are described, in for example Holliger & Hudson,Nature Biotechnology 23(9):1126-1136 (2005).

In some preferred embodiments, the anti-exosite 1 antibody molecule maybe a whole antibody. For example, the anti-exosite 1 antibody moleculemay be an IgG, IgA, IgE or IgM or any of the isotype sub-classes,particularly IgG1 and IgG4. The anti-exosite 1 antibody molecules may bemonoclonal antibodies. In other preferred embodiments, the anti-exosite1 antibody molecule may be an antibody fragment.

Anti-exosite 1 antibody molecules may be chimeric, humanised or humanantibodies.

Anti-exosite 1 antibody molecules as described herein may be isolated,in the sense of being free from contaminants, such as antibodies able tobind other polypeptides and/or serum components. Monoclonal antibodiesare preferred for some purposes, though polyclonal antibodies may alsobe employed.

Anti-exosite 1 antibody molecules may be obtained using techniques whichare standard in the art. Methods of producing antibodies includeimmunising a mammal (e.g. mouse, rat, rabbit, horse, goat, sheep ormonkey) with the protein or a fragment thereof. Antibodies may beobtained from immunised animals using any of a variety of techniquesknown in the art, and screened, preferably using binding of antibody toantigen of interest. For instance, Western blotting techniques orimmunoprecipitation may be used (Armitage et al., 1992, Nature 357:80-82). Isolation of antibodies and/or antibody-producing cells from ananimal may be accompanied by a step of sacrificing the animal.

As an alternative or supplement to immunising a mammal with a peptide,an antibody specific for a protein may be obtained from a recombinantlyproduced library of expressed immunoglobulin variable domains, e.g.using lambda bacteriophage or filamentous bacteriophage which displayfunctional immunoglobulin binding domains on their surfaces; forinstance see WO92/01047. The library may be naive, that is constructedfrom sequences obtained from an organism which has not been immunisedwith any of the proteins (or fragments), or may be one constructed usingsequences obtained from an organism which has been exposed to theantigen of interest.

Other anti-exosite 1 antibody molecules may be identified by screeningpatient serum for antibodies which bind to exosite 1.

In some embodiments, anti-thrombin antibody molecules may be produced byany convenient means, for example a method described above, and thenscreened for differential binding to mature thrombin relative tothrombin with an exosite 1 mutation, gamma thrombin (exosite 1 defectivedue to autolysis at R75 and R77a) or prothrombin. Suitable screeningmethods are well-known in the art.

An antibody which displays increased binding to mature thrombin,relative to non-thrombin proteins, thrombin with an exosite 1 mutation,gamma-thrombin or prothrombin, for example an antibody which binds tomature thrombin but does not bind to thrombin with an exosite Imutation, gamma thrombin or prothrombin, may be identified as ananti-exosite 1 antibody molecule.

After production and/or isolation, the biological activity of ananti-exosite 1 antibody molecule may be tested. For example, the abilityof the antibody molecule to inhibit thrombin substrate, cofactor orinhibitor binding and/or cleavage by thrombin may be determined and/orthe ability of the antibody molecule to inhibit thrombosis withoutpromoting bleeding may be determined.

Suitable antibody molecules may be tested for activity using afibrinogen clotting or thrombin time assay. Suitable assays arewell-known in the art.

The effect of an antibody molecule on coagulation and bleeding may bedetermined using standard techniques. For example, the effect of anantibody molecule on thrombosis may be determined in an animal model,such as a mouse model with ferric chloride induced clots in bloodvessels. Effects on haemostasis may also be determined in an animalmodel, for example, by measuring tail bleed of a mouse.

Antibody molecules normally comprise an antigen binding domaincomprising an immunoglobulin heavy chain variable domain (VH) and animmunoglobulin light chain variable domain (VL), although antigenbinding domains comprising only a heavy chain variable domain (VH) arealso possible (e.g. camelid or shark antibodies).

Each of the VH and VL domains typically comprise three complementaritydetermining regions (CDRs) responsible for antigen binding, interspersedby framework regions.

In some embodiments, binding to exosite 1 may occur wholly orsubstantially through the VHCDR3 of the anti-exosite 1 antibodymolecule.

For example, an anti-exosite 1 antibody molecule may comprise a VHdomain comprising a HCDR3 having the amino acid sequence of SEQ ID NO: 5or the sequence of SEQ ID NO: 5 with 1 or more, for example 2, 3, 4 or 5or more amino acid substitutions, deletions or insertions. Thesubstitutions may be conservative substitutions. In some embodiments,the HCDR3 may comprise the amino acid residues at positions 4 to 9 ofSEQ ID NO: 5 (SEFEPF), or more preferably the amino acid residues atpositions 2, and 4 to 10 of SEQ ID NO: 5 (D and SEFEPFS) withsubstitutions, deletions or insertions at one or more other positions inSEQ ID NO:5. The HCDR3 may be the only region of the antibody moleculethat interacts with a thrombin exosite 1 epitope or substantially theonly region. The HCDR3 may therefore determine the specificity and/oraffinity of the antibody molecule for the exosite 1 region of thrombin.

The VH domain of an anti-exosite 1 antibody molecule may additionallycomprise an HCDR2 having the amino acid sequence of SEQ ID NO: 4 or thesequence of SEQ ID NO: 4 with 1 or more, for example 2, 3, 4 or 5 ormore amino acid substitutions, deletions or insertions. In someembodiments, the HCDR2 may comprise the amino acid residues at positions3 to 7 of SEQ ID NO: 4 (DPQDG) or the amino acid residues at positions 2and 4 to 7 of SEQ ID NO: 4 (L and PQDG) of SEQ ID NO: 4, withsubstitutions, deletions or insertions at one or more other positions inSEQ ID NO: 4.

The VH domain of an anti-exosite 1 antibody molecule may furthercomprise an HCDR1 having the amino acid sequence of SEQ ID NO: 3 or thesequence of SEQ ID NO: 3 with 1 or more, for example 2, 3, 4 or 5 ormore amino acid substitutions, deletions or insertions. In someembodiments, the HCDR1 may comprise amino acid residue T at position 5of SEQ ID NO: 3 with substitutions, deletions or insertions at one ormore other positions in SEQ ID NO: 3.

In some embodiments, an antibody molecule may comprise a VH domaincomprising a HCDR1, a HCDR2 and a HCDR3 having the sequences of SEQ IDNOs 3, 4 and 5 respectively. For example, an antibody molecule maycomprise a VH domain having the sequence of SEQ ID NO: 2 or the sequenceof SEQ ID NO: 2 with 1 or more, for example 2, 3, 4, 5, 6, 7, 8, 9, 10or more amino acid substitutions, deletions or insertions in SEQ ID NO:2.

The anti-exosite 1 antibody molecule may further comprise a VL domain,for example a VL domain comprising LCDR1, LCDR2 and LCDR3 having thesequences of SEQ ID NOs 7, 8 and 9 respectively, or the sequences of SEQID NOs 7, 8 and 9 respectively with, independently, 1 or more, forexample 2, 3, 4 or 5 or more amino acid substitutions, deletions orinsertions. The substitutions may be conservative substitutions. Forexample, an antibody molecule may comprise a VL domain having thesequence of SEQ ID NO: 6 or the sequence of SEQ ID NO: 6 with 1 or more,for example 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions,deletions or insertions in SEQ ID NO: 6.

In some embodiments, the VL domain may comprise Tyr49.

The anti-exosite 1 antibody molecule may for example comprise one ormore amino acid substitutions, deletions or insertions which improve oneor more properties of the antibody, for example affinity, functionalhalf-life, on and off rates.

The techniques that are required in order to introduce substitutions,deletions or insertions within amino acid sequences of CDRs, antibody VHor VL domains and antibodies are generally available in the art. Variantsequences may be made, with substitutions, deletions or insertions thatmay or may not be predicted to have a minimal or beneficial effect onactivity, and tested for ability to bind exosite 1 of thrombin and/orfor any other desired property.

In some embodiments, anti-exosite 1 antibody molecule may comprise a VHdomain comprising a HCDR1, a HCDR2 and a HCDR3 having the sequences ofSEQ ID NOs 3, 4, and 5, respectively, and a VL domain comprising aLCDR1, a LCDR2 and a LCDR3 having the sequences of SEQ ID NOs 7, 8 and9, respectively.

For example, the VH and VL domains may have the amino acid sequences ofSEQ ID NO: 2 and SEQ ID NO: 6 respectively; or may have the amino acidsequences of SEQ ID NO: 2 and SEQ ID NO: 6 comprising, independently 1or more, for example 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acidsubstitutions, deletions or insertions. The substitutions may beconservative substitutions.

In some embodiments, an antibody may comprise one or more substitutions,deletions or insertions which remove a glycosylation site. For example,a glycosylation site in VL domain of SEQ ID NO 6 may be mutated out byintroducing a substitution at either N28 or S30.

The anti-exosite 1 antibody molecule may be in any format, as describedabove, In some preferred embodiments, the anti-exosite 1 antibodymolecule may be a whole antibody, for example an IgG, such as IgG1 orIgG4, IgA, IgE or IgM.

An anti-exosite 1 antibody molecule of the invention may be one whichcompetes for binding to exosite 1 with an antibody molecule describedabove, for example an antibody molecule which

-   -   (i) binds thrombin exosite 1 and    -   (ii) comprises a VH domain of SEQ ID NO: 2 and/or VL domain of        SEQ ID NO: 6; an HCDR3 of SEQ ID NO: 5; an HCDR1, HCDR2, LCDR1,        LCDR2, or LCDR3 of SEQ ID NOS: 3, 4, 7, 8 or 9 respectively; a        VH domain comprising HCDR1, HCDR2 and HCDR3 sequences of SEQ ID        NOS: 3, 4 and 5 respectively; and/or a VH domain comprising        HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOS: 3, 4 and 5 and a        VL domain comprising LCDR1, LDR2 and LCDR3 sequences of SEQ ID        NOS: 7, 8 and 9 respectively.

Competition between antibody molecules may be assayed easily in vitro,for example using ELISA and/or by tagging a specific reporter moleculeto one antibody molecule which can be detected in the presence of one ormore other untagged antibody molecules, to enable identification ofantibody molecules which bind the same epitope or an overlappingepitope. Such methods are readily known to one of ordinary skill in theart. Thus, a further aspect of the present invention provides anantibody molecule comprising a antibody antigen-binding site thatcompetes with an antibody molecule, for example an antibody moleculecomprising a VH and/or VL domain, CDR e.g. HCDR3 or set of CDRs of theparent antibody described above for binding to exosite 1 of thrombin. Asuitable antibody molecule may comprise an antibody antigen-binding sitewhich competes with an antibody antigen-binding site for binding toexosite 1 wherein the antibody antigen-binding site is composed of a VHdomain and a VL domain, and wherein the VH and VL domains compriseHCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOS: 3, 4, and 5 and LCDR1,LDR2 and LCDR3 sequences of SEQ ID NOS: 7, 8, and 9 respectively, forexample the VH and VL domains of SEQ ID NOS: 2 and 6.

An anti-exosite 1 antibody molecule as described herein may inhibit thebinding of thrombin-binding factors, including factors which bind toexosite 1. For example, an antibody molecule may competitively ornon-competitively inhibit the binding of one or more of fV, fVIII,thrombomodulin, fibrinogen or fibrin, PAR1 and/or hirugen and hirudinanalogues to thrombin.

An anti-exosite 1 antibody molecule as described herein may inhibit oneor more activities of thrombin. For example, an anti-exosite 1 antibodymolecule may inhibit the hydrolytic cleavage of one or more thrombinsubstrates, such as fibrinogen, platelet receptor PAR-1 and coagulationfactor FVIII. For example, binding of the antibody molecule to thrombinmay result in an at least 5-fold, at least 10-fold, or at least 15-folddecrease in the hydrolysis of fibrinogen, PAR-1, coagulation factorFVIII and/or another thrombin substrates, such as factor V, factor XIIIin the presence of fibrin, and protein C and/or TAFI in the presence ofthrombomodulin. In some embodiments, binding of thrombin by theanti-exosite 1 antibody molecule may result in no detectable cleavage ofthe thrombin substrate by thrombin.

Techniques for measuring thrombin activity, for example by measuring thehydrolysis of thrombin substrates in vitro are standard in the art andare described herein.

Anti-exosite 1 antibody molecules may be further modified by chemicalmodification, for example by PEGylation, or by incorporation in aliposome, to improve their pharmaceutical properties, for example byincreasing in vivo half-life.

The effect of an anti-exosite 1 antibody molecule on coagulation andbleeding may be determined using standard techniques. For example, theeffect of an antibody on a thrombosis model may be determined. Suitablemodels include ferric chloride clot induction in blood vessels in amurine model, followed by a tail bleed to test normal haemostasis. Othersuitable thrombosis models are well known in the art (see for exampleWestrick et al ATVB (2007) 27:2079-2093)

Anti-exosite 1 antibody molecules may be comprised in pharmaceuticalcompositions with a pharmaceutically acceptable excipient.

A pharmaceutically acceptable excipient may be a compound or acombination of compounds entering into a pharmaceutical compositionwhich does not provoke secondary reactions and which allows, forexample, facilitation of the administration of the anti-exosite 1antibody molecule, an increase in its lifespan and/or in its efficacy inthe body or an increase in its solubility in solution. Thesepharmaceutically acceptable vehicles are well known and will be adaptedby the person skilled in the art as a function of the mode ofadministration of the anti-exosite 1 antibody molecule.

In some embodiments, anti-exosite 1 antibody molecules may be providedin a lyophilised form for reconstitution prior to administration. Forexample, lyophilised antibody molecules may be re-constituted in sterilewater and mixed with saline prior to administration to an individual.

Anti-exosite 1 antibody molecules will usually be administered in theform of a pharmaceutical composition, which may comprise at least onecomponent in addition to the antibody molecule. Thus pharmaceuticalcompositions may comprise, in addition to the anti-exosite 1 antibodymolecule, a pharmaceutically acceptable excipient, carrier, buffer,stabilizer or other materials well known to those skilled in the art.Such materials should be non-toxic and should not interfere with theefficacy of the anti-exosite 1 antibody molecule. The precise nature ofthe carrier or other material will depend on the route ofadministration, which may be by bolus, infusion, injection or any othersuitable route, as discussed below.

For parenteral, for example sub-cutaneous or intra-venousadministration, e.g. by injection, the pharmaceutical compositioncomprising the anti-exosite 1 antibody molecule may be in the form of aparenterally acceptable aqueous solution which is pyrogen-free and hassuitable pH, isotonicity and stability. Those of relevant skill in theart are well able to prepare suitable solutions using, for example,isotonic vehicles, such as Sodium Chloride Injection, Ringer'sInjection, Lactated Ringer's Injection. Preservatives, stabilizers,buffers, antioxidants and/or other additives may be employed as requiredincluding buffers such as phosphate, citrate and other organic acids;antioxidants, such as ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride; benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens, such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3′-pentanol; and m-cresol); low molecularweight polypeptides; proteins, such as serum albumin, gelatin orimmunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone;amino acids, such as glycine, glutamine, asparagines, histidine,arginine, or lysine; monosaccharides, disaccharides and othercarbohydrates including glucose, mannose or dextrins; chelating agents,such as EDTA; sugars, such as sucrose, mannitol, trehalose or sorbitol;salt-forming counter-ions, such as sodium; metal complexes (e.g.Zn-protein complexes); and/or non-ionic surfactants, such as TWEEN™,PLURONICS™ or polyethylene glycol (PEG).

A pharmaceutical composition comprising an anti-exosite 1 antibodymolecule may be administered alone or in combination with othertreatments, either simultaneously or sequentially dependent upon thecondition to be treated.

An anti-exosite 1 antibody molecule as described herein may be used in amethod of treatment of the human or animal body, including prophylacticor preventative treatment (e.g. treatment before the onset of acondition in an individual to reduce the risk of the condition occurringin the individual; delay its onset; or reduce its severity after onset).The method of treatment may comprise administering an anti-exosite 1antibody molecule to an individual in need thereof.

Administration is normally in a “therapeutically effective amount”, thisbeing sufficient to show benefit to a patient. Such benefit may be atleast amelioration of at least one symptom. The actual amountadministered, and rate and time-course of administration, will depend onthe nature and severity of what is being treated, the particular mammalbeing treated, the clinical condition of the individual patient, thecause of the disorder, the site of delivery of the composition, themethod of administration, the scheduling of administration and otherfactors known to medical practitioners. Prescription of treatment, e.g.decisions on dosage etc, is within the responsibility of generalpractitioners and other medical doctors and may depend on the severityof the symptoms and/or progression of a disease being treated.Appropriate doses of antibody molecules are well known in the art(Ledermann J. A. et al. (1991) Int. J. Cancer 47: 659-664; Bagshawe K.D. et al. (1991) Antibody, Immunoconjugates and Radiopharmaceuticals 4:915-922). Specific dosages may be indicated herein or in the Physician'sDesk Reference (2003) as appropriate for the type of medicament beingadministered may be used. A therapeutically effective amount or suitabledose of an antibody molecule may be determined by comparing its in vitroactivity and in vivo activity in an animal model. Methods forextrapolation of effective dosages in mice and other test animals tohumans are known. The precise dose will depend upon a number of factors,including whether the antibody is for prevention or for treatment, thesize and location of the area to be treated, the precise nature of theantibody (e.g. whole antibody, fragment) and the nature of anydetectable label or other molecule attached to the antibody.

A typical antibody dose will be in the range 100 μg to 1 g for systemicapplications, and 1 μg to 1 mg for topical applications. An initialhigher loading dose, followed by one or more lower doses, may beadministered. Typically, the antibody will be a whole antibody, e.g. theIgG1 or IgG4 isotype. This is a dose for a single treatment of an adultpatient, which may be proportionally adjusted for children and infants,and also adjusted for other antibody formats in proportion to molecularweight. Treatments may be repeated at daily, twice-weekly, weekly ormonthly intervals, at the discretion of the physician. The treatmentschedule for an individual may be dependent on the pharmocokinetic andpharmacodynamic properties of the antibody composition, the route ofadministration and the nature of the condition being treated.

Treatment may be periodic, and the period between administrations may beabout two weeks or more, e.g. about three weeks or more, about fourweeks or more, about once a month or more, about five weeks or more, orabout six weeks or more. For example, treatment may be every two to fourweeks or every four to eight weeks. Treatment may be given before,and/or after surgery, and/or may be administered or applied directly atthe anatomical site of surgical treatment or invasive procedure.Suitable formulations and routes of administration are described above.

In some embodiments, anti-exosite 1 antibody molecules as describedherein may be administered as sub-cutaneous injections. Sub-cutaneousinjections may be administered using an auto-injector, for example forlong term prophylaxis/treatment.

In some preferred embodiments, the therapeutic effect of theanti-exosite 1 antibody molecule may persist for several half-lives,depending on the dose. For example, the therapeutic effect of a singledose of anti-exosite 1 antibody molecule may persist in an individualfor 1 month or more, 2 months or more, 3 months or more, 4 months ormore, 5 months or more, or 6 months or more.

Anti-exosite 1 antibody molecules described herein inhibit thrombin andmay be useful in the treatment of thrombin-mediated conditions.

Haemostasis is the normal coagulation response i.e. the prevention ofbleeding or haemorrhage, for example from a damaged blood vessel.Haemostasis arrests bleeding and haemorrhage from blood vessels in thebody.

Anti-exosite 1 antibody molecules may have no effect or substantially noeffect on haemostasis i.e. they do not promote bleeding or haemorrhage.

Aspects of the invention provide; an anti-exosite 1 antibody molecule asdescribed herein for use in a method of treatment of the human or animalbody; an anti-exosite 1 antibody molecule as described herein for use ina method of treatment of a thrombin-mediated disorder; the use of ananti-exosite 1 antibody molecule as described herein in the manufactureof a medicament for the treatment of a thrombin-mediated condition; anda method of treatment of a thrombin-mediated condition comprisingadministering an anti-exosite 1 antibody molecule as described herein toan individual in need thereof.

Inhibition of thrombin by anti-exosite 1 antibodies as described hereinmay be of clinical benefit in the treatment of any thrombin-mediatedcondition. A thrombin-mediated condition may include disordersassociated with the formation or activity of thrombin.

Thrombin plays a key role in haemostasis, coagulation and thrombosis.Thrombin-mediated conditions include thrombotic conditions, such asthrombosis and embolism.

Thrombosis is coagulation which is in excess of what is required forhaemostasis (i.e. excessive coagulation), or which is not required forhaemostasis (i.e. extra-haemostatic or non-haemostatic coagulation).

Thrombosis is blood clotting within the blood vessel lumen. It ischaracterised by the formation of a clot (thrombus) that is in excess ofrequirement or not required for haemostasis. The clot may impede bloodflow through the blood vessel leading to medical complications. A clotmay break away from its site of formation, leading to embolism elsewherein the circulatory system. In the arterial system, thrombosis istypically the result of atherosclerotic plaque rupture.

In some embodiments, thrombosis may occur after an initial physiologicalhaemostatic response, for example damage to endothelial cells in a bloodvessel. In other embodiments, thrombosis may occur in the absence of anyphysiological haemostatic response.

Thrombosis may occur in individuals with an intrinsic tendency tothrombosis (i.e. thrombophilia) or in ‘normal’ individuals with nointrinsic tendency to thrombosis, for example in response to anextrinsic stimulus.

Thrombosis and embolism may occur in any vein, artery or other bloodvessel within the circulatory system and may include microvascularthrombosis.

Thrombosis and embolism may be associated with surgery (either duringsurgery or afterwards) or the insertion of foreign objects, such ascoronary stents, into a patient.

For example, anti-exosite 1 antibodies as described herein may be usefulin the surgical and other procedures in which blood is exposed toartificial surfaces, such as open heart surgery and dialysis.

Thrombotic conditions may include thrombophilia, thrombotic stroke andcoronary artery occlusion.

Patients suitable for treatment as described herein include patientswith conditions in which thrombosis is a symptom or a side-effect oftreatment or which confer an increased risk of thrombosis or patientswho are predisposed to or at increased risk of thrombosis, relative tothe general population. For example, an anti-exosite 1 antibody moleculeas described herein may also be useful in the treatment or prevention ofvenous thrombosis in cancer patients, and in the treatment or preventionof hospital-acquired thrombosis, which is responsible for 50% of casesof venous thromboembolism.

Anti-exosite 1 antibody molecules as described herein may exert atherapeutic or other beneficial effect on thrombin-mediated conditions,such as thrombotic conditions, without substantially inhibiting orimpeding haemostasis. For example, the risk of haemorrhage in patientstreated with anti-exosite 1 antibody molecules may not be increased orsubstantially increased relative to untreated individuals.

Individuals treated with conventional anticoagulants, such as naturaland synthetic heparins, warfarin, direct serine protease inhibitors(e.g. argatroban, dabigatran, apixaban, and rivaroxaban), hirudin andits derivatives (e.g. lepirudin and bivalirudin), and anti-plateletdrugs (e.g. clopidogrel, ticlopidine and abciximab) cause bleeding. Therisk of bleeding in patients treated with anti-exosite 1 antibodymolecules as described herein may be reduced relative to individualstreated with conventional anticoagulants.

Thrombin-mediated conditions include non-thrombotic conditionsassociated with thrombin activity, including inflammation, infection,tumour growth and metastasis, organ rejection and dementia (vascular andnon-vascular, e.g. Alzheimer's disease) (Licari et al J Vet Emerg CritCare (San Antonio). 2009 February; 19(1):11-22; Tsopanoglou et al EurCytokine Netw. 2009 Dec. 1; 20(4):171-9).

Anti-exosite 1 antibody molecules as described herein may also be usefulin in vitro testing, for example in the analysis and characterisation ofcoagulation, for example in a sample obtained from a patient.

Anti-exosite 1 antibody molecules may be useful in the measurement ofthrombin generation. Assays of thrombin generation are technicallyproblematic because the conversion of fibrinogen to fibrin causesturbidity, which precludes the use of a simple chromogenic end-point.

The addition of an anti-exosite 1 antibody molecule as described hereinto a sample of blood prevents or inhibits fibrin formation and henceturbidity and permits thrombin generation to be measured using achromogenic substrate, without the need for a defibrination step.

For example, a method of measuring thrombin generation may comprisecontacting a blood sample with a chromogenic thrombin substrate in thepresence of an anti-exosite 1 antibody molecule as described herein andmeasuring the chromogenic signal from the substrate;

-   -   wherein the chromogenic signal is indicative of thrombin        generation in the sample.

The chromogenic signal may be measured directly without defibrination ofthe sample.

Suitable substrates are well known in the art and include S2238(H-D-Phe-Pip-Arg-pNa), β-Ala-Gly-Arg-p-nitroanilide diacetate (Prasa, D.et al. (1997) Thromb. Haemost. 78, 1215; Sigma Aldrich Inc) andTos-Gly-Pro-Arg-pNa.AcOH (Biophen CS-01(81); Aniara Inc OH USA).

Anti-exosite 1 antibody molecules may also be useful in inhibiting orpreventing the coagulation of blood as described above in extracorporealcirculations, such as haemodialysis and extracorporeal membraneoxygenation.

For example, a method of inhibiting or preventing blood coagulation invitro or ex vivo may comprise introducing an anti-exosite 1 antibodymolecule as described herein to a blood sample. The blood sample may beintroduced into an extracorporeal circulation system before,simultaneous with or after the introduction of the anti-exosite 1antibody and optionally subjected to treatment such as haemodialysis oroxygenation. In some embodiments, the treated blood may be subsequentlyadministered to an individual. Other embodiments provide an anti-exosite1 antibody molecule as described herein for use in a method ofinhibiting or preventing blood coagulation in a blood sample ex vivo andthe use of an anti-exosite 1 antibody molecule as described herein inthe manufacture of a medicament for use in a method of inhibiting orpreventing blood coagulation in a blood sample ex vivo.

Other aspects of the invention relate to the production of antibodymolecules which bind to the exosite 1 epitope of thrombin and may beuseful, for example in the treatment of pathological blood coagulationor thrombosis.

A method for producing an antibody antigen-binding domain for theexosite 1 epitope of thrombin, may comprise;

-   -   providing, by way of addition, deletion, substitution or        insertion of one or more amino acids in the amino acid sequence        of a parent VH domain comprising HCDR1, HCDR2 and HCDR3, wherein        HCDR1, HCDR2 and HCDR3 have the amino acid sequences of SEQ ID        NOS: 3, 4 and 5 respectively, a VH domain which is an amino acid        sequence variant of the parent VH domain, and;    -   optionally combining the VH domain thus provided with one or        more VL domains to provide one or more VH/VL combinations; and    -   testing said VH domain which is an amino acid sequence variant        of the parent VH domain or the VH/VL combination or combinations        to identify an antibody antigen binding domain for the exosite 1        epitope of thrombin.

A VH domain which is an amino acid sequence variant of the parent VHdomain may have the HCDR3 sequence of SEQ ID NO: 5 or a variant with theaddition, deletion, substitution or insertion of one, two, three or moreamino acids.

The VH domain which is an amino acid sequence variant of the parent VHdomain may have the HCDR1 and HCDR2 sequences of SEQ ID NOS: 3 and 4respectively, or variants of these sequences with the addition,deletion, substitution or insertion of one, two, three or more aminoacids.

A method for producing an antibody molecule that specifically binds tothe exosite 1 epitope of thrombin may comprise:

-   -   providing starting nucleic acid encoding a VH domain or a        starting repertoire of nucleic acids each encoding a VH domain,        wherein the VH domain or VH domains either comprise a HCDR1,        HCDR2 and/or HCDR3 to be replaced or lack a HCDR1, HCDR2 and/or        HCDR3 encoding region;    -   combining said starting nucleic acid or starting repertoire with        donor nucleic acid or donor nucleic acids encoding or produced        by mutation of the amino acid sequence of an HCDR1, HCDR2,        and/or HCDR3 having the amino acid sequences of SEQ ID NOS: 3, 4        and 5 respectively, such that said donor nucleic acid is or        donor nucleic acids are inserted into the CDR1, CDR2 and/or CDR3        region in the starting nucleic acid or starting repertoire, so        as to provide a product repertoire of nucleic acids encoding VH        domains;    -   expressing the nucleic acids of said product repertoire to        produce product VH domains;    -   optionally combining said product VH domains with one or more VL        domains;    -   selecting an antibody molecule that binds exosite 1 of thrombin,        which antibody molecule comprises a product VH domain and        optionally a VL domain; and    -   recovering said antibody molecule or nucleic acid encoding it.

Suitable techniques for the maturation and optimisation of antibodymolecules are well-known in the art.

Antibody antigen-binding domains and antibody molecules for the exosite1 epitope of thrombin may be tested as described above. For example, theability to bind to thrombin and/or inhibit the cleavage of thrombinsubstrates may be determined.

The effect of an antibody molecule on coagulation and bleeding may bedetermined using standard techniques. For example, a mouse thrombosismodel of ferric chloride clot induction in a blood vessel, such as thefemoral vein or carotid artery, followed by a tail bleed to test normalhaemostasis, may be employed.

Various further aspects and embodiments of the present invention will beapparent to those skilled in the art in view of the present disclosure.

All documents mentioned in this specification are incorporated herein byreference in their entirety.

Unless stated otherwise, antibody residues are numbered herein inaccordance with the Kabat numbering scheme.

“and/or” where used herein is to be taken as specific disclosure of eachof the two specified features or components with or without the other.For example “A and/or B” is to be taken as specific disclosure of eachof (i) A, (ii) B and (iii) A and B, just as if each is set outindividually herein.

Unless context dictates otherwise, the descriptions and definitions ofthe features set out above are not limited to any particular aspect orembodiment of the invention and apply equally to all aspects andembodiments which are described. Thus, the features set out above aredisclosed in all combinations and permutations.

Certain aspects and embodiments of the invention will now be illustratedby way of example and with reference to the figures and tables describedbelow.

FIG. 1 shows the binding and elution of the IgA on humanthrombin-Sepharose column. FIG. 1A shows an elution profile for IgA(narrow peak) from a thrombin-Sepharose column using a pH gradient(neutral to low, indicated by upward sloping line). FIG. 1B shows anative blue gel showing total IgA load, flow-through from the humanthrombin column and eluate following elution at low pH.

FIG. 2 shows a non-reducing SDS-PAGE gel which indicates that the IgAbinds thrombin but not prothrombin. In this pull-down assay, lectinagarose is used to bind to IgA in the presence of thrombin orprothrombin. The supernatant is then run on an SDS gel. Lane 1 is sizestandards; lane 2 shows a depletion of thrombin from the supernatant;Lane 3 shows that depletion is dependent on the presence of the IgA;Lanes 3 and 4 show that prothrombin is not depleted, and therefore doesnot bind to the IgA.

FIG. 3 shows the relative rate of S2238 cleavage by thrombin in thepresence or absence of IgA (i.e. a single slope of Abs405 with time forS2238 hydrolysis). This indicates that the IgA does not bind at thethrombin active site.

FIG. 4 shows the results of binding studies which indicate that the IgAcompetes with the fluorescently labelled dodecapeptide hirugen forbinding to thrombin.

FIG. 5 shows the effect of the IgA on the cleavage of S2238 by thrombin.This analysis allows the estimate of Kd for the IgA-thrombin interactionof 12 nM.

FIG. 6 shows an SDS-PAGE gel of whole IgA and Fab fragments underreducing and non-reducing (ox) conditions. The non-reduced IgA is shownto have a molecular weight of between 100-200 kDa and the non-reducedFab has a molecular weight of about 50 kDa.

FIG. 7 shows the crystal structure of Thrombin-Fab complex showinginteraction between the exosite 1 of thrombin and HCDR3 of the Fabfragment.

FIG. 8 shows detail of crystal structure showing interaction betweenspecific residues of thrombin exosite 1 and HCDR3 of the Fab fragment.

FIG. 9 shows fluorescence microscopy images of FeCl₃ induced blood clotsin femoral vein injuries in C57BL/6 mice injected with FITC labelledfibrinogen taken at between 2 and 30 minutes. 100 ul of PBS wasadministered (vehicle control).

FIG. 10 shows fluorescence microscopy images of FeCl₃ induced bloodclots in femoral vein injuries in C57BL/6 mice injected with FITClabelled fibrinogen and 40 nM (final concentration in mouse blood,equivalent to a dose of approximately 0.6 mg/Kg) anti-exosite 1 IgA (100μl in PBS).

FIG. 11 shows fluorescence microscopy images of FeCl₃ induced bloodclots in femoral vein injuries in C57BL/6 mice injected with FITClabelled fibrinogen and 80 nM (final concentration in mouse blood,equivalent to a dose of approximately 1.2 mg/Kg) anti-exosite 1 IgA(100μl in PBS), and a region outside of injury site for comparison.

FIG. 12 shows fluorescence microscopy images of FeCl₃ induced bloodclots in femoral vein injuries in C57BL/6 mice injected with FITClabelled fibrinogen and 200 nM (final concentration in mouse blood,equivalent to a dose of approximately 3 mg/Kg) anti-exosite 1 IgA (100μl in PBS), and a region outside of injury site for comparison.

FIG. 13 shows fluorescence microscopy images of FeCl₃ induced bloodclots in femoral vein injuries in C57BL/6 mice injected with FITClabelled fibrinogen and 400 nM (final concentration in mouse blood,equivalent to a dose of approximately 6 mg/Kg) anti-exosite 1 IgA (100μl in PBS).

FIG. 14 shows fluorescence microscopy images of FeCl₃ induced bloodclots in femoral vein injuries in C57BL/6 mice treated with FITClabelled fibrinogen and 4 μM (final concentration in mouse blood,equivalent to a dose of approximately 60 mg/Kg) anti-exosite 1 IgA (100μl in PBS).

FIG. 15 shows a quantitation of the dose response to anti-exosite 1 IgAfrom the fluorescent images shown in FIGS. 9 to 13.

FIG. 16 shows tail bleed times in control C57BL/6 mice and in micetreated with increasing amounts of anti-exosite 1 IgA. The secondaverage excludes the outlier.

FIG. 17 shows the results of tail clip assays on wild-type male C57BL/6mice (n=5) after injection into tail vein with either IgA or PBS. 15 minafter injection, tails were cut at diameter of 3 mm and blood lossmonitored over 10 min.

FIG. 18A to 18D show the results of an FeCl₃ carotid artery occlusionmodel on 9 week old WT C57BL/6 male mice injected as previously with 400nM anti-thrombin IgA (final concentration in blood, equivalent to a doseof approximately 6 mg/Kg) or PBS 15 min prior to injury with 5% FeCl₃for 2 min. FIG. 18A shows results for a typical PBS-injected mice(occlusion in 20 min) and FIGS. 18B, 18C and 18D show examples ofresults for mice treated with 400 nM anti-thrombin IgA (no occlusion).

FIG. 19 shows thrombin times (i.e. clotting of pooled plasma) withincreasing concentrations of IgG and IgA of the invention, upon additionof 20 nM human thrombin.

FIG. 20 shows the binding of synthetic IgG to immobilized thrombin (onForteBio Octet Red instrument).

FIG. 21 shows a typical Octet trace for the binding of 24 nM S195Athrombin to immobilized IgG showing the on phase, followed by an offphase. The black line is the fit.

FIG. 22 shows an Octet trace of 500 nM prothrombin with a tip loadedwith immobilized IgG. The same conditions were used as the experimentwith thrombin in FIG. 21. There is no evidence of binding, even at thishigh concentration.

FIG. 23 shows the ELISA binding curves for anti-exosite 1 IgG and an IgGS30A variant binding to thrombin.

FIG. 24 shows the potency of IgG and IgG S30A in an ex vivo activatedpartial thromboplastin time (APTT) coagulation assay.

FIG. 25 shows time to stop bleeding for 30 seconds data for IgG S30A andIgG in the rat tail clip bleeding model.

FIG. 26 shows total bleeding time data for IgG S30A and IgG in the rattail clip bleeding model.

FIG. 27 shows total hemoglobin lost data for IgG S30A and IgG in the rattail clip bleeding model.

FIG. 28 shows data on the prevention of thrombus formation by IgG S30Aand IgG in the rat venous thrombosis model using ferric chloride (FeCl₃)at 2.5% concentration.

FIG. 29 shows data on the prevention of thrombus formation by IgG S30Aand IgG in the rat venous thrombosis model using ferric chloride (FeCl₃)at 5% concentration.

EXPERIMENTS

1. Antibody Isolation and Characterisation

Coagulation screening was carried out on a blood plasma sample from apatient. The coagulation tests were performed on a patient who sufferedsubdural haematoma following head injury. The haematoma spontaneouslyresolved without intervention. There was no previous history of bleedingand in the 4 years since the patient presented, there have been nofurther bleeding episodes. The results are shown in Table 1.

The prothrombin time (PT), activated partial thromboplastin time (APTT),and thrombin time (TT) were all prolonged in the patient compared tocontrols, but reptilase time was normal.

Thrombin time was not corrected by heparinase, indicating that heparintreatment or contamination was not responsible. Fibrinogen levels werenormal in the patient, according to ELISA and Reptilase assays. TheClauss assay gave an artifactally low fibrinogen level due to thepresence of the thrombin inhibitor. The PT and APTT clotting times werefound to remain prolonged following a mixing test using a 50:50 mix withpooled plasma from normal individuals. This showed the presence of aninhibitor in the sample from the patient.

The patient's blood plasma was found to have a high titre of an IgA.This IgA molecule was found to bind to a human thrombin column (FIG. 1).IgA binding lectin-agarose pulled down thrombin in the presence but notthe absence of the IgA. Prothrombin was not pulled down by thelectin-agarose in the presence of the IgA, indicating that the IgAspecifically binds to thrombin but not prothrombin (FIG. 2).

The binding site of the IgA on the thrombin molecule was theninvestigated.

A slightly higher rate of cleavage of S2238 by thrombin was measured inthe presence of the IgA, indicating that the IgA does not block theactive site of thrombin (FIG. 3).

The binding of fluorescently labelled hirugen to thrombin is inhibitedby the presence of 700 nM of the IgA, indicating that the epitope forthe antibody overlaps with the binding site of hirugen on thrombin,namely the exosite 1 of thrombin (FIG. 4).

The effect of the IgA on the hydrolysis of some of thrombin'sprocoagulant substrates was tested. The results are shown in Table 2.These results demonstrate that the IgA molecule isolated from thepatient sample inhibits multiple procoagulant activities of thrombin.

Inhibition of thrombin by antithrombin (AT) in the presence of the IgAwas only marginally affected in both the absence and presence of heparin(Table 3).

The dissociation constant (K_(d)) of the IgA for thrombin was initiallyestimated based on rate of S2238 hydrolysis to be approximately 12 nM(FIG. 5). The K_(d) for the binding of the IgA to S195A thrombin(inactivated by mutation of the catalytic serine) was determined to be 2nM using the ForteBio Octet Red instrument (Table 4).

The purified IgA was cleaved with papain (FIG. 6), and the Fab fragmentwas isolated and combined with human PPACK-Thrombin (PPACK is a covalentactive site inhibitor). The human PPACK-Thrombin-FAB complex wascrystallized and used for structural analysis. The statistics of thestructure obtained were as follows: resolution is 1.9 Å; Rfactor=19.43%;Rfree=23.42%; one complex in the asymmetric unit; Ramachandran:favoured=97.0%, outliers=0%. The crystal structure revealed a closeassociation between the HCDR3 of the IgA Fab and the exosite 1 ofthrombin (FIG. 7).

In particular, residues M32, F34, Q38, E39, L40, L65, R67, R73, T74,R75, Y76, R77a and 182 of the exosite 1 all directly interact with theHCDR3 loop of the IgA Fab (FIG. 8).

PISA analysis of the antibody-thrombin interface showed that the totalburied surface area in the complex is 1075 Å². The contact residues inthe IgA heavy chain were (Kabat numbering): 30, 51, 52a, 53-55, 96, 98,99, 100, 100a, 100b, 100c, 100d). These are all in CDRs:CDRH1-GYTLTEAAIH; CDRH2-GLDPQDGETVYAQQFKG; CDRH3-GDFSEFEPFSMDYFHF(underlined residues contacting). CDRH3 was found to be the mostimportant, providing 85% of the buried surface area on the antibody. Thelight chain made one marginal contact with Tyr49, right before CDRL2(with Ser36a of thrombin). Some individual contributions to buriedsurface were: Glu99 54 Å², Phe100 134.8 Å², Glu100a 80.6 Å², Phe100c141.7 Å².

The contact residues in thrombin were found to be (chymotrypsinnumbering): 32, 34, 36a-40, 65, 67, 73-76, 77a, 82, and 151. The mostimportant individual contributors to the buried surface were: Gln38 86.4Å², Arg73 44.5 Å², Thr74 60.1 Å², Tyr76 78.4 Å², Arg77a 86.9 Å².

The patient did not display increased or abnormal bleeding orhaemorrhage, in spite of 3 g/l circulating levels of this IgA,demonstrating that the antibody inhibits thrombin without affectingnormal haemostasis.

2. The Effect of IgA on Animal Thrombosis Models

C57BL/6 mice were anaesthetized. A catheter was inserted in the carotidartery (for compound injection). FITC labelled fibrinogen (2 mg/ml) wasinjected via the carotid artery. PBS (control) or IgA was also injectedvia the carotid artery. The femoral vein was exposed and 10% FeCl₃applied (saturated blotting paper 3 mm in length) for 3 min to induceclotting.

Fluorescence microscopy images were taken along the length of injurysite at 0, 5, 10, and 20 min post FeCl₃ injury using fluorescencemicroscopy techniques.

Clots (fibrin deposits) in the femoral vein were clearly visible asbright areas (FIG. 9). The lowest dose of the antibody was observed tocause significant inhibition of clotting but as the dose increased,clotting was abolished (FIGS. 10 to 15).

The bleeding times of the mice were also measured. Bleeding times wereassessed as time to cessation of blood flow after a tail cut. Despitethe presence of a single outlier sample, the bleeding time was found tobe unaffected by treatment with anti-exosite 1 IgA (FIG. 16).

These results show that the anti-exosite 1 IgA antibody is a potentinhibitor of thrombosis but has no effect on bleeding time.

3. Tail Clip Assays

A tail clip assay was performed on wild-type male C57BL/6 mice injectedwith either 400 nM IgA (final concentration in blood, equivalent to adose of approximately 6 mg/Kg) or PBS. Blood loss was monitored over 10mins after the tail was cut at 3 mm diameter 15 minutes after theinjection. Total blood loss was found to be unaffected by treatment withanti-exosite 1 IgA (FIG. 17).

4. FeCl₃ Injury Carotid Artery Occlusion

FeCl₃ injury carotid artery occlusion studies were performed on 9 weekold WT C57BL/6 male mice. Mice were injected with 400 nM anti-IIa IgA(final concentration in blood, equivalent to a dose of approximately 6mg/Kg) or PBS 15 min prior to injury with 5% FeCl₃ for 2 min. Blood flowwas then monitored by Doppler and the time to occlusion measured. A“clot” was defined as stable occlusive thrombus where blood flow wasreduced to values typically less than 0.1 ml/min and stayed reduced. Inthe control mice, a stable clot was observed to form about 20 mins afterinjury (FIG. 18A). However, the majority of mice treated with 400 nManti-IIa IgA were unable to form stable clots and gave traces in whichthe clots were quickly resolved, repeatedly resolved or never formed.Three representative traces are shown in FIGS. 18B to 18D.

5. Anti-Exosite 1 IgG

The IgA molecule identified in the patient described above wasre-formatted as an IgG using standard techniques.

The clotting time of pooled human plasma spiked with increasing amountsof the original IgA and the new IgG was tested upon addition of humanthrombin to 20 nM (FIG. 19). Both parent IgA and the synthetic IgGincreased time to clot formation in an identical concentration-dependentmanner, implying identical affinities for thrombin.

This was confirmed by measuring the binding of synthetic IgG toimmobilized S195A thrombin using a ForteBio™ Octet Red instrument.Thrombin was attached to the probe and the binding of the antibodies (atvarious concentrations) was monitored. On-rates and off-rates weredetermined. Both antibodies gave similar on-rates of approximately 3×10⁵M⁻¹s⁻¹ and off-rates of approximately 5×10⁻⁴ s⁻¹, and dissociationconstants (Kd) of approximately 2 nM. Kds of approximately 2 nM werealso obtained for the IgA and the IgG by steady-state analysis (Table4). A representative steady state curve is shown in FIG. 20. Theproperties of the IgA were therefore reproduced on an IgG framework.

Binding of prothrombin to the IgG antibody was tested using the Octetsystem by immobilizing IgG. Thrombin bound to the immobilized IgG withcomparable rates and affinities as those obtained using immobilizedthrombin (Table 4); prothrombin did not bind to the IgG. FIG. 21 is atrace of 24 nM thrombin binding to and dissociating from the immobilizedIgG. FIG. 22 is the same experiment using 500 nM prothrombin, and showsno evidence of binding.

6. Anti-Exosite 1 IgG S30A Variant Antibody

6.1 Introduction

Glycosylation sites in an antibody can raise issues during manufactureand/or therapeutic use of the antibody. The oligosaccharides added toglycosylation sites are typically heterogenous, for example with complexdi-antenary and hybrid oligosaccharides with sialic acids and galactoses(for Fab oligosaccharides) or with fucosylated non-galactosylateddi-antenary oligosaccharides (for Fc oligosaccharides). The presence ofmore than one glycosylation site in an antibody (or active fragmentthereof) thus adds further to potential heterogeneity. Removal ofincorrectly glycosylated forms of an antibody during the purificationprocess is very difficult and can lead to extended process developmentactivities and reduced yields.

Therefore, if a glycosylation site in an antibody (or active fragmentthereof) is determined not to be required directly or indirectly forantigen binding activity, it may be desirable from a manufacturing andquality control perspective to remove that glycosylation site byengineering.

As noted above, it was envisaged that a glycosylation site in VL domainof SEQ ID NO 6 of the antibody of the present invention could be mutatedout by introducing a substitution at either N28 or 530.

Of the two residues N28 and 530, S30 was targeted for substitution as itwas considered, based on crystal structure analysis, less likely to beinvolved in antibody folding or stability.

6.2 Methods and Materials

An “IgG S30A” variant monoclonal antibody was produced using standardsite-directed mutagenesis techniques from the anti-exosite IgG antibody(“IgG”) described in section 5 above by substituting serine residue 30(S30) with an alanine (hence, S30A).

The IgG S30A variant was expressed for analysis using standard transientexpression techniques as described below.

In outline, single gene vectors (SGVs) were constructed using GS Xceedvectors (Lonza Biologics, Slough, UK) (pXC IgG4pro ΔK for the heavychain constant domain encoding region and pXC Kappa for light chainconstant domain encoding region) and the variable domain encodingregions as synthesised by GeneArt AG. The SGVs were amplified andtransiently co-transfected into Chinese Hamster Ovary CHOK1SV GS KOcells for initial expression at a volume of 200 ml and then subsequentlyat a scaled-up volume of 2.5 litres.

The methods used will be described below. Where manufacturer'sinstructions were followed, this will be indicated.

6.2.1 Vector Construction

The sequences of the light and heavy chain variable domain encodingregions were synthesised by GeneArt AG. Light chain variable domainencoding region was sub-cloned into pXC Kappa and heavy chain variabledomain encoding region into pXC IgG4pro ΔK vectors respectively usingthe N-terminal restriction site Hind III and the C-terminal restrictionsites BsiWI (light chain) and ApaI (heavy chain). In short, the 5 μl oflyophilised shuttle vectors, as produced by GeneArt AG, were resuspendedin 50 μl endotoxin free, sterile water. 1 μg of DNA was digested withthe relevant restriction enzymes in a total volume of 50 μl and sampleswere incubated for 2 hours at 37° C. 8.3 μl of 6×DNA loading buffer wasadded and samples electrophoresed at 120 V for 40 min on a 1% w/vagarose gel stained with ethidium bromide. 10 μl Lonza SimplyLoad TandemDNA ladder was used as reference ladder.

The relevant fragments were gel-extracted using a QIAquick gelextraction kit (QIAGEN, 28704) according to a manufacturer'sinstructions. Ligations were set-up using Roche's quick ligation kitwith a 1:12 ratio of vector backbone to insert DNA, 1 μl T4 quickligase, 10 μl of 2×T4 quick ligation buffer, reaction volume adjusted to21 μl with endotoxin-free, sterile water when necessary and samplesincubated at room temperature for 10 minutes. 10 μl aliquots of theligation reactions were used to transform One Shot Top 10 ChemicallyCompetent Escherichia coli cells (Invitrogen, C404003) using theheat-shock method according to manufacturer's instructions. Cells werespread onto ampicillin-containing (50 μg/ml) Luria Bertani agar plates(LB Agar, Sigma-Aldrich L7025) and incubated overnight at 37° C. untilbacterial colonies were evident. Positive clones were screened by PCRamplification and verified by restriction digest (using a double digestof EcoRI-HF and HindIII-HF) and nucleotide sequencing of the gene ofinterest through a 3^(rd) party provider.

6.2.2 DNA Amplification

A single bacterial colony was picked into 15 ml Luria Bertani (LB)medium (LB Broth, Sigma-Aldrich, L7275) containing 50 μl/ml ampicillinand incubated at 37° C. overnight with shaking at 220 rpm. The resultingstarter culture was used to inoculate 1 L Luria Bertani (LB) mediumcontaining 50 μl/mg ampicillin and incubated at 37° C. overnight withshaking at 220 rpm. Vector DNA was isolated using the QIAGEN PlasmidPlus Gigaprep system (QIAGEN, 12991). In all instances, DNAconcentration was measured using a Nanodrop 1000 spectrophotometer(Thermo-Scientific) and adjusted to 1 mg/ml with EB buffer (10 mMTris-Cl, pH 8.5).

6.2.3 Routine Culture of CHOK1SV GS KO Cells

CHOK1SV GS KO cells were cultured in CD-CHO media (Invitrogen 10743-029)supplemented with 6 mM glutamine (Invitrogen, 25030-123). Cells wereincubated in a shaking incubator at 36.5° C., 5% CO₂, 85% humidity, 140rpm. Cells were routinely sub-cultured every 3-4 days, sending at 2×10⁵cells/ml and were propagated in order to have sufficient cells availablefor transfection. Cells were discarded by passage 20.

6.2.4 Transient Transfections of CHOK1SV GS KO Cells

Transient transfections were performed using CHOK1SV GS KO cells whichhad been in culture a minimum two weeks. Cells were sub-cultured 24 hprior to transfection.

All transfections were carried out via electroporation using either theGene Pulse XCell (Bio-Rad), a cuvette based electroporation system forsmall scale (200 ml) transfections or a Gene Pulse MXCell (Bio-Rad), aplate based system for electroporation for the larger scale (2.5 L)transfection. For each transfection, viable cells were resuspended inpre-warmed media to 2.86×10⁷ cells/ml. 80 μg DNA (1:1 ratio of heavy andlight chain SGVs) and 700 μl cell suspension were aliquotted into eachcuvette/well. Cells were electroporated at 300 V, 900 μF for the GenePulse XCell system and 300 V, 1300 μF for the Gene Pulse MXCell system.Transfected cells were transferred to pre-warmed media in Erlenmeyerflasks and the cuvette/wells rinsed twice with pre-warmed media whichwas also transferred to the flasks. Transfected cell cultures wereincubated in a shaking incubator at 36.5° C., 5% CO₂, 85% humidity, 140rpm for 6 days. Cell viability and viable cell concentrations weremeasured at the time of harvest using a Cedex HiRes automated cellcounter (Roche).

6.2.5 Protein A Affinity Chromatography

Small (200 ml) and large (2.5 L) scale culture supernatant wereharvested and clarified by centrifugation at 2000 rpm for 10 min, thenfiltered through a 0.22 μm filter. Clarified supernatant was purifiedusing a pre-packed 5 ml HiTrap MabSelect SuRE column (GE Healthcare,11-0034-94) on an AKTA purifier (10 ml/min). The column was equilibratedwith 50 mM sodium phosphate, 125 mM sodium chloride, pH 7.0(equilibration buffer) for 5 column volumes (CVs). After sample loading,the column was washed with 2 CVs of equilibration buffer followed by 3CVs of 50 mM sodium phosphate, 1 M sodium chloride pH 7.0 and a repeatwash of 2 CVs of equilibration buffer. The Product was then eluted with10 mM sodium formate, pH 3.5 over 5 CVs. Protein containing, elutedfractions were immediately pH adjusted to pH 7.2 and filtered through a0.2 μm filter.

6.2.6 SE-HPLC Analysis

Duplicate samples were analysed to SE-HPLC on an Agilent 1200 seriesHPLC system, using a Zorbax GF-250 4 μm 9.4 mm ID×250 mm column(Agilent). Aliquots of sample at a concentration of 1 mg/ml werefiltered through a 0.2 μm filter prior to injection. 80 μl aliquots wereinjected respectively and run at 1 ml/min for 15 minutes. Solubleaggregate levels were analysed using Chemstation (Agilent) software.

6.2.7 SDS-PAGE Analysis

Reduced samples were prepared for analysis by mixing with NuPage 4×LDSsample buffer (Invitrogen, NP0007) and NuPage 10× sample reducing agent(Invitrogen NP0009), and incubated at 70° C., 10 min. For non-reducedsamples, the reducing agent and heat incubation were omitted. Sampleswere electrophoresed on 1.5 mm NuPage 4-12% Bis-Tris Novex pre-cast gels(Invitrogen, NP0335PK2) with NuPage MES SDS running buffer underdenaturing conditions. 10 μl aliquots of SeeBlue Plus 2 pre-stainedmolecular weight standard (Invitrogen, LC5925) and a control IgG4antibody at 1 mg/ml were included on the gel. 1 μl of each sample at 1mg/ml were loaded onto the gel. Once electrophoresed, gels were stainedwith InstantBlue (TripleRed, ISB01L) for 30 min at room temperature.Images of the stained gels were analysed on a BioSpectrum Imagine System(UVP).

6.2.7 Endotoxin Analysis

Endotoxin levels purified protein from the larger scale (2.5 L)production was measured at 2.54 mg/ml using the Endosafe-PTS instrument,a cartridge based method based on the LAL assay (Charles River).

6.3 Results and Discussion

The transfectant culture from the initial expression at a volume of 200ml was harvested on Day 6 post-transfection and clarified bycentrifugation and sterile filtration. The clarified cell culturesupernatant was purified using one-step Protein A chromatography.Quantification was by absorbance at A_(280nm). Production qualityanalysis in the form of SE-HPLC and SDS-PAGE showed a high level ofpurity was achieved post-purification.

For scaling up the culture volume up to 2.5 litres, as before, Day 6harvested, clarified cell culture supernatant was purified usingone-step Protein A chromatography. Product quality analysis in the formof SE-HPLC, SDS-PAGE and endotoxin detection was carried out usingpurified material at a concentration of 1 mg/ml, alongside an in-househuman IgG4 antibody as a control sample. High level of purity wasobserved from the purified ichorcumab S30A with a small trace of highmolecular weight impurity (1.8%) and an endotoxin level below thedetectable scale of <0.02 EU/mg.

Thereafter, analysis of the IgG S30A variant produced as above wasperformed using standard techniques to check in vitro and in vivoactivity compared with the anti-exosite IgG antibody.

FIG. 23 shows that IgG S30A has equivalent or higher binding affinity tothrombin than the IgG antibody, as determined by a standard ELISAbinding assay.

Using a standard ex vivo activated partial thromboplastin time (APTT)coagulation assay, IgG S30A was found to be equivalent or more potentthan IgG.

Table 5 shows IgG and IgG S30A binding affinities to thrombin usingBiacorem surface binding analysis (GE Healthcare, Little Chalfont,Buckinghamshire, UK). IgG S30A has equivalent or higher affinity tothrombin compared to IgG. Affinities were not affected for either IgGS30A or IgG by storage for one month at 4° C. or by exposure to light(PO).

Table 6 shows that both IgG S30A and IgG have equivalent solubility andboth are soluble to >100 mg/ml concentration, with little reduction insolubility (and no aggregate formation) on storage.

FIG. 24 shows the potency of IgG and IgG S30A in an ex vivo activatedpartial thromboplastin time (APTT) coagulation assay. IgG S30A isequivalent or more potent than IgG.

FIG. 25 shows that both IgG S30A and IgG are equivalent in the rat tailclip bleeding model (see experimental section 3 above), with bothshowing no difference to vehicle control in time to stop bleeding for 30seconds.

FIG. 26 shows that both IgG S30A and IgG are equivalent in the rat tailclip bleeding model, with both showing no difference to vehicle controlin total bleeding time.

FIG. 27 shows that both IgG S30A and IgG are equivalent in the rat tailclip bleeding model, with both showing no difference to vehicle controlin total hemoglobin lost.

FIG. 28 shows that both IgG S30A and IgG are equivalent in the ratvenous thrombosis model using ferric chloride (FeCl₃; see experimentalsection 2 above) at 2.5% concentration, with both IgG S30A and IgGcausing total prevention of thrombus formation.

FIG. 29 shows that both IgG S30A and IgG are equivalent in the ratvenous thrombosis model using ferric chloride (FeCl₃) at 5%concentration, with both IgG S30A and IgG causing similar reduction ofthrombus formation.

The results showed that the removal of the S30 glycosylation site in theIgG antibody to form the IgG S30A variant did not negatively impact onthe binding or other beneficial characteristics of the antibody. The IgGS30A variant thus may be preferable from a manufacturing and productionperspective for reasons described above.

Specific anti-exosite 1 antibody molecules disclosed herein include thefollowing:

(1) a wild-type anti-exosite 1 IgA antibody;

(2) a synthetic anti-exosite 1 IgG antibody (also referred to herein as“IgG”), re-formatted from the wild-type IgA antibody; and

(3) a synthetic anti-exosite 1 IgG S30A variant antibody (also referredto herein as “IgG S30A”), which compared with the IgG antibody above hasan S30A substitution.

The IgG antibody has the wild-type sequence of IgA in the VH and VLdomains. The IgG S30A antibody has the wild type sequence of IgA and IgGin the VH and VL domains, except that a glycosylation site in VL domainof SEQ ID NO 6 has been mutated out by introducing a substitution(alanine for serine) at 530.

In the specific examples, the synthetic monoclonal antibodies IgG andIgG S30A are also referred to by the name “ichorcumab”.

7. Large-scale production of IgG S30A variant antibody

7.1 Introduction

In experimental section 6 above, the IgG S30A variant was expressedtransiently using standard techniques for the purposes of analysing thevariant. Here, we show that large scale production of IgG S30A followingstable cell transfection using standard techniques is also possible.

7.2 Materials and Methods

In outline, double gene vector (DGV) was constructed using previouslyestablished single gene vectors (see experimental section 6 above) inLonza's GS Xceed vectors (pXC IgG4pro ΔK for the heavy chain constantdomain encoding region and pXC Kappa for light chain constant domainencoding region). The DGV was amplified and stably transfected intoCHOK1SV GS-KO cells and analysed.

The methods used will be described below. Where manufacturer'sinstructions were followed, this will be indicated.

7.2.1 Vector Construction

Single gene vectors (SGVs) established in Lonza's GS Xceed vectors fromthe previous transient production of ichorcumab S30A (see experimentalsection 6 above) were used to generate a double gene vector (DGV). TheDGV was constructed by restriction digest of the established SGVs usingPvul (Roche, 10650129001) and NotI (Roche, 11014714001) in a totalreaction volume of 20 μl and incubated at 37° C. for 2 hours. 4.0 μl of6×DNA loading buffer was added to the digested samples andelectrophoresed at 120 V for 40 min on a 1% w/v agarose gel stained withethidium bromide. 10 μl Lonza SimplyLoad Tandem DNA ladder was used as areference ladder. The agarose gel was imaged using BioSpectrum ImagingSystem (IVP).

The relevant fragments were gel-extracted using a QIAquick gelextraction kit (QIAGEN, 28704) according to manufacturer's instructions.Ligations were set-up using Roche's quick ligation kit (Roche,11635379001) with a 1:3 ratio of vector backbone to insert DNA, 1 μl T4quick ligase, 10 μl of 2×T4 quick ligation buffer, 2 μl of 10×DNAdilution buffer, reaction volume adjusted to 21 μl with endotoxin-free,sterile water when necessary and samples incubated at room temperaturefor 10 minutes. 10 μl aliquots of the ligation reactions were used totransform One Shot Top 10 Chemically Competent Escherichia coli cells(Invitrogen, C404003) using the heat-shock method according tomanufacturer's instructions.

Cells were spread onto ampicillin-containing (50 μg/ml) APS Media (APSLB Broth base, BD 292438) agar plates and incubated overnight at 37° C.until bacterial colonies were evident. Positive clones were screened byPCT amplification and verified by restriction digest (using aHindIII/EcoRI double restriction digest) and nucleotide sequencing ofthe coding regions through a 3^(rd) party provider.

7.2.2 DNA Amplification

For DNA amplification, 5 ml of the growth cultures produced during thecolony screening were used to inoculate 1 L APS medium (APS LB Brothbase, BD 292438) containing 50 μg/ml ampicillin, incubated at 37° C.overnight and shaking at 220 rpm. Vector DNA was isolated using theQIAGEN Plasmid Plus Gigaprep system (QIAGEN, 12991) and quantified usinga Nanodrop 1000 spectrophotometer (Thermo-Scientific).

7.2.3 Routine Culture of CHOK1SV GS-KO Cells

CHOK1SV GS-KO cells were cultured in CD-CHO media (Invitrogen,10743-029) supplemented with 6 mM L-glutamine (Invitrogen, 25030-123).Cells were incubated in a shaking incubator at 36.5° C., 5% CO₂, 85%humidity, 140 rpm. Cells were routinely sub-cultured every 3-4 days,seeding at 2×10⁵ cells/ml and were propagated in order to havesufficient cells available for transfection. Cells were discarded bypassage 20.

7.2.4 Stable Pooled Transfection of CHOK1SV GS-KO Cells

Double gene vector DNA plasmids were prepared for transfection bylinearising with Pvul followed by ethanol precipitation and resuspensionin EB buffer to a final concentration of 400 μg/ml. Transfections werecarried out via electroporation using either the Gene Pulse XCell(Bio-Rad). For each transfection, viable cells were resuspended in apre-warmed CD-CHO media to 1.43×10⁷ cells/ml. 100 μl linearised DNA at aconcentration of 400 μg/ml was aliquotted into a 0.4 cm gapelectroporation cuvette and 700 μl cell suspension added. Three cuvettesof cells and DNA were electroporated at 300 V, 900 μF and immediatelyrecovered to 30 ml pre-warmed CD-CHO supplemented with 10 ml/L SP4(Lonza, BESP1076E) to generate a stable pool. The transfectants wereincubated in a shaking incubator at 36.5° C., 5% CO₂, 85% humidity, 140rpm.

A total of 5 stable pool transfectants were established. 24 hpost-transfection the cultures were centrigued and resuspended intopre-warmed CD-CHO supplemented with 50 μM MSX (L-Methionine Sulfoximine,Sigma-Aldrich, M5379) and 10 ml/L SP4. Cell growth and viability wereperiodically checked post-transfection.

When the viable cell density reached >0.6×10⁵ cells/ml, the transfectantcultures were suitable to process. Cells were seeded at 0.2×10⁶ cells/mlin a final volume of 100 ml in CD-CHO medium supplemented with 50 μMMSX/10 ml/L SP4, in a 500 ml vented Erlenmeyer flask (Fisher Scientific(Corning), 10352742) and incubated in a shaking incubator at 36.5° C.,5% CO₂, 85% humidity, 140 rpm. Cell cultures were monitored and expandedonce cultures had adapted to exponential growth. Cultures were thenexpanded to the appropriate production volume.

7.2.5 Protein A HPLC

Duplicate samples of clarified cell culture supernatant were analysed byProtein A HPLC on an Agilent 1200 series HPLC system, using a POROSProtein A cartridge (Applied Biosystems, 2-1001-00). 100 μl aliquots ofsupernatant samples, 0.22 μm filtered, were injected and run in 50 mMglycine, 150 mM sodium chloride, pH 8.0 at 2 ml/min for 5 minuteseluting with 50 mM glycine, 150 mM sodium chloride, pH 2.5. An 8-pointstandard curve was generated with 2-fold dilutions of a 1 mg/ml IgG₄in-house standard. All sample chromatograms were analysed usingChemstation software.

7.2.6 Cryopreservation of Cells

Five (5) vials each of the top two producing stable pools, as screenedby Protein A HPLC during the suspension adaptation phase, werecryopreserved. Each vial contains 1.5 ml cell culture at 1×10⁷ cells/ml,passage number 3, with viability in excess of 98% prior tocryopreservation. Cells were centrifuged at 900 rpm for 5 minutes, thesupernatant discarded and the cell pellet resuspended in ambient CD-CHOsupplemented with 7.5% v/v DMSO. The vials were transferred into a Mr.Frosty™ (ThermoFisher) to −80° C. before the frozen vials weretransferred into vapour phase nitrogen storage.

7.2.7 Abridged Fed-Batch Overgrow Study

Cells were propagated to production volume by seeding the appropriateculture at 0.2×10⁶ cells/ml in Lonza's CM42 base media supplemented with4 ml/L SPE using the established stable pools. The production volume wasestablished in 5 L shake flasks (Generon, 931116). Shake flask cultureswere incubated in a shaking incubator at 36.5° C., 5% CO₂, 85% humidity,and 140 rpm. Two batches of culture were initiated with a preliminary 1L culture to deduce production titre followed by a 40 L productioninitiated one week later. Cell count and viability were monitored on day4, before feeding was initiated, and periodically until the culture washarvested on day 12. The bolus feeds were administered on day 4 and 8consisting of a mixture of Lonza's proprietary feeds.

7.2.8 Harvesting and Concentrating of Production Culture

2.9 L of the 40 L production culture was harvested by centrifugation at6000 rpm prior to depth filtration using a KLEENPAK nova cartridge(PALL, NT6UBP1G), followed by filter sterilisation using a KLEENPAK 0.22μm filter cartridge (PALL, KA2EKVP1G). The remaining supernatant wascentrifuged as above and subject to clarification using pilot scalesystems. The supernatant was frozen and stored at −20° C.

7.2.9 Protein A Affinity Chromatography

Clarified supernatant was purified using a 100 ml HiTrap MabSelect SuREcolumn (GE Healthcare, 17-5438-02) on an AKTA purifier (20 ml/min). Thecolumn was equilibrated with 50 mM sodium phosphate, 125 mM sodiumchloride, pH 7.0 (equilibration buffer) for 5 column volumes (CVs).After sample loading, the column was washed with 2 CVs of equilibrationbuffer followed by 3 CVs of 50 mM sodium phosphate, 1 M sodium chloridepH 7.0 and a repeat wash of 2 CVs of equilibration buffer. The productwas then eluted with 10 mM sodium formate, pH 3.5 over 5 CVs. Proteincontaining, eluted fractions were immediately pH adjusted to pH 7.2 andfiltered through a 0.2 μm filter.

7.2.10 SE-HPLC Analysis

Duplicate samples were analysed by SE-HPLC on an Agilent 1200 seriesHPLC system, using a Zorbax GF-250 4 μm 9.4 mm ID×250 mm column(Agilent). Aliquots of sample at a concentration of 1 mg/ml werefiltered through a 0.2 μm filter prior to injection. 80 μl aliquots wereinjected respectively and run at 1 ml/min for 15 minutes. Solubleaggregate levels were analysed using Chemstation (Agilent) software.

7.2.11 SDS-PAGE Analysis

Reduced samples were prepared for analysis by mixing with NuPage 4×LDSsample buffer (Invitrogen, NP0007) and NuPage 10× sample reducing agent(Invitrogen, NP0009), and incubated at 70° C., 10 min. For non-reducedsamples, the reducing agent and heat incubation were omitted. Sampleswere electrophoresed on 1.5 mm NuPage 4-12% Bis-Tris Novex pre-cast gels(Invitrogen, NP0335PK2) with NuPage MES SDS running buffer underdenaturing conditions. 10 μl aliquots of SeeBlue Plus 2 pre-stainedmolecular weight standard (Invitrogen, LC5925) and a control IgG₄antibody at 1 mg/ml were included on the gel. 1 μl of each sample at 1mg/ml were loaded onto the gel. Once electrophoresed, gels were stainedwith InstantBlue (TripleRed, ISB01L) for 30 min at room temperature.Images of the stained gels were analysed on a BioSpectrum Imaging System(UVP).

7.2.12 Endotoxin Analysis

Endotoxin levels of the purified product were tested once concentratingto 20 mg/ml was completed. The product was tested at 1 mg/ml using theEndosafe-PTS instrument, a cartridge based method based on the LAL assay(Charles River).

7.3 Results and Discussion

Initially, 5 stable pools of transfectant cultures were produced. Thetransfectant cultures were screened by Protein A HPLC to identify thetop 2 expressing pools. A 1 L preliminary culture followed by a 40 Lproduction culture were initiated and subjected to an abridged fed-batchovergrow study including the administration of bolus feeds on days 4 and8. Cultures were harvested on Day 12 and supernatant titre determinedprior to harvest. A volume of the sample culture was clarified bycentrifugation followed by depth and sterile filtration. The clarifiedcell culture supernatant was purified using one-step Protein Achromatography.

Product quality analysis in the form of SE-HPLC, SDS-PAGE and endotoxindetection showed a high level of purity was achieved post-purification.The remaining supernatant was clarified using a pilot scale filtrationsystem due to high viscosity and large amount of product present withinthe supernatant.

SEQUENCES

Amino acid sequence of human preprothrombin (SEQ ID NO: 1; GeneID: 2147;NP 000497.1 GI: 4503635; exosite 1 residues underlined)

1 mahvrglqlp gclalaalcs lvhsqhvfla pqqarsllqr vrrantflee vrkgnlerec 61veetcsyeea fealesstat dvfwakytac etartprdkl aaclegncae glgtnyrghv 121nitrsgiecq lwrsryphkp einstthpga dlqenfcrnp dssttgpwcy ttdptvrrqe 181csipvcgqdq vtvamtprse gssvnlsppl eqcvpdrgqq yqgrlavtth glpclawasa 241qakalskhqd fnsavqlven fcrnpdgdee gvwcyvagkp gdfgycdlny ceeaveeetg 301dgldedsdra iegrtatsey qtffnprtfg sgeadcglrp lfekksledk terellesyi 361dgrivegsda eigmspwqvm lfrkspqell cgaslisdrw vltaahclly ppwdknften 421dllvrigkhs rtryerniek ismlekiyih prynwrenld rdialmklkk pvafsdyihp 481vclpdretaa sllqagykgr vtgwgnlket wtanvgkgqp svlqvvnlpi verpvckdst 541riritdnmfc agykpdegkr gdacegdsgg pfvmkspfnn rwyqmgivsw gegcdrdgky 601gfythvfrlk kwiqkvidqf ge

Amino acid sequence of anti-exosite 1 IgA and IgG VH domain with KabatNumbering (CDRs underlined): (SEQ ID NO: 2).

QVQLIQSGSAVKKPGASVRVSCKVSGYTLTEAAIHWVRQAPGKGLEWMGG        10        20        30        40        50LDPQDGETVYAQQFKGRVTMTEDRSTDTAYMEVNNLRSEDTATYYCTTGD52a      60        70        8082abc      90 FSEFEPFSMDYFHFWGQGTVVTVAS 100abcdefgh       110

Amino acid sequence of anti-exosite 1 IgA and IgG HCDR1 (SEQ ID NO: 3).

GYTLTEAAIH

Amino acid sequence of anti-exosite 1 IgA and IgG HCDR2 (SEQ ID NO: 4).

GLDPQDGETVYAQQFKG

Amino acid sequence of anti-exosite 1 IgA and IgG HCDR3 (SEQ ID NO: 5).

GDFSEFEPFSMDYFHF

Amino acid sequence of anti-exosite 1 IgA and IgG VL domain with KabatNumbering: (SEQ ID NO: 6).

EIVLTQSPATLSLSPGERATLSCRASQNVSSFLAWYQHKPGQAPRLLIYD        10        20        30        40        50ASSRATDIPIRFSGSGSGTDFTLTISGLEPEDFAVYYCQQRRSWPPLTFG          60        70        80        90   95a GGTKVEIKR  100     108

Amino acid sequence of anti-exosite 1 IgA and IgG LCDR1 (SEQ ID NO: 7).

RASQNVSSFLA

Amino acid sequence of anti-exosite 1 IgA and IgG LCDR2 (SEQ ID NO: 8).

DASSRAT

Amino acid sequence of anti-exosite 1 IgA and IgG LCDR3 (SEQ ID NO: 9).

QQRRSWPPLT

TABLE 1 Coagulation Screening Results Control/Normalised Test ResultRatio (NR) Prothrombin Time  43 sec. NR = 11-13 sec. 50:50  35 sec.correction Act. part. 157 sec. NR = 22-23 sec. Thromboplastin Time 50:50105 sec. correction Thrombin Time >150 sec.   NR = 10-13 sec. ReptilaseTime  16 sec. Control = 15 sec. Fibrinogen Clauss 0.7 g/l NR = 1.5-4.5g/l Antigenic 5.0 g/l

TABLE 2 Effect of anti-exosite 1 IgA on thrombin hydrolysis ofprocoagulant substrates Thrombin substrate Activity Antibody EffectFibrinogen Formation of fibrin No detectable clot cleavage Plateletreceptor Activation of 15-fold decrease in PAR-1 peptide plateletshydrolysis FVIII Feedback activation 7-fold decrease in of thrombin viahydrolysis Xase complex

TABLE 3 Effect of saturating concentration of anti-exosite 1 IgA (Fab)on thrombin inhibition by antithrombin (AT) in the absence and presenceof 1 nM heparin (Hep). Rate of Inhibition(M⁻¹s⁻¹) Heparin effect AT 4.8± 0.2 × 10³ 2.4-fold AT + Hep 11.8 ± 0.3 × 10³  AT + Fab 1.7 ± 0.1 × 10³3.3-fold AT + Hep + Fab 5.6 ± 0.3 × 10³

TABLE 4 Binding constants of anti-exosite 1 IgA (n = 1 under thisprecise condition), IgG (n = 3) antibodies, and IgG-derived FAB to S195Athrombin (active site free, recombinant thrombin). Kd (nM)* k_(on)(M⁻¹s⁻¹) k_(off) (s⁻¹) Kd (nM)# IgA 1.8 3.3 × 10⁵ 3.7 × 10⁻⁴ 1.2 IgG 1.5± 0.3 3.3 ± 0.5 × 10⁵ 6.8 ± 1.1 × 10⁻⁴ 2.1 ± 0.3 IgG FAB ND 5.0 × 10⁵2.7 × 10⁻³ 5.3 IgG FAB⁺ 3.3 ± 0.3 4.3 × 10⁵ 2.1 × 10⁻³ 4.9 *Kddetermined from steady-state analysis of response vs. concentration. #Kdcalculated from rates. ⁺Determined using immobilised FAB.

TABLE 5 Binding affinities of IgG and IgG S30A to thrombin usingBiacore ™ surface binding analysis. Binding at ambient condition(“Control”) was compared with binding (1) after storage for one month at4° C. or (2) after exposure to light (“PO”). Ligand Kd (nM) IgG S30AControl 1.77 IgG S30A 4° C. 1.74 IgG S30A PO 1.80 IgG Control 4.08 IgG4° C. 3.95 IgG PO 4.00

TABLE 6 Solubility of IgG S30A and IgG (in mg/ml) and affect of storage(at 4° C.) IgG S30A IgG Time (mg/mL) (mg/mL) Day 0 128 ± 0 129 ± 0 Day 6120 ± 0 122 ± 0 Day 28 113 ± 1 123 ± 2

1-27. (canceled)
 28. An isolated or recombinant nucleic acid moleculeencoding an antibody molecule that specifically binds to the exosite 1region of thrombin, comprising an LCDR1 having the amino acid sequenceof SEQ ID NO: 15, an LCDR2 having the amino acid sequence of SEQ ID NO:8, and an LCDR3 having the amino acid sequence of SEQ ID NO: 9, andcomprising an HCDR1 having the amino acid sequence of SEQ ID NO: 3, anHCDR2 having the amino acid sequence of SEQ ID NO: 4, and an HCDR3having the amino acid sequence of SEQ ID NO:
 5. 29. The nucleic acidmolecule according to claim 28, wherein the antibody molecule comprisesa variable heavy chain region (VH) having the amino acid sequence of SEQID NO:
 2. 30. The nucleic acid molecule according to claim 28, whereinthe antibody molecule comprises a variable light chain region (VL)having the amino acid sequence of SEQ ID NO:
 14. 31. A vector orrecombinant cell, comprising the nucleic acid molecule of claim
 28. 32.A method of producing an antibody molecule that specifically binds tothe exosite 1 region of thrombin, comprising culturing the recombinantcell according to claim 31 under conditions such that the antibodymolecule is expressed and recovered.
 33. The nucleic acid moleculeaccording to claim 28, wherein the antibody molecule is a Fab, diabody,triabody, tetrabody, or minibody.
 34. The nucleic acid moleculeaccording to claim 28, wherein the antibody molecule is a monoclonalantibody.
 35. The nucleic acid molecule according to claim 33, whereinthe monoclonal antibody comprises an IgG constant region.
 36. Thenucleic acid molecule according to claim 34, wherein the IgG constantregion is an IgG1 or IgG4 constant region.
 37. The nucleic acid moleculeaccording to claim 28, wherein the antibody molecule is a humanantibody.
 38. An isolated or recombinant nucleic acid molecule encodingan antibody molecule that specifically binds to the exosite 1 region ofthrombin, comprising a variable light chain region (VL) comprising theamino acid sequence of SEQ ID NO: 14 and a variable heavy chain region(VH) comprising an HCDR1 having the amino acid sequence of SEQ ID NO: 3,an HCDR2 having the amino acid sequence of SEQ ID NO: 4, and an HCDR3having the amino acid sequence of SEQ ID NO:
 5. 39. The nucleic acidmolecule according to claim 38, wherein the antibody molecule is amonoclonal antibody.
 40. The nucleic acid molecule according to claim39, wherein the monoclonal antibody comprises an IgG constant region.41. The nucleic acid molecule according to claim 40, wherein the IgGconstant region is an IgG1 or IgG4 constant region.
 42. The nucleic acidmolecule according to claim 38, wherein the antibody molecule is a humanantibody.
 43. A vector or recombinant cell, comprising the nucleic acidmolecule of claim
 38. 44. A method of producing an antibody moleculethat specifically binds to the exosite 1 region of thrombin, comprisingculturing the recombinant cell according to claim 43 under conditionssuch that the antibody molecule is expressed and recovered.
 45. Anisolated or recombinant nucleic acid molecule encoding an antibodymolecule, comprising a variable light chain region (VL) comprising theamino acid sequence of SEQ ID NO: 14 and a variable heavy chain region(VH) comprising the amino acid sequence of SEQ ID NO:
 2. 46. The nucleicacid molecule according to claim 45, wherein the antibody molecule is amonoclonal antibody.
 47. The nucleic acid molecule according to claim46, wherein the monoclonal antibody comprises an IgG constant region.48. The nucleic acid molecule according to claim 47, wherein the IgGconstant region is an IgG1 or IgG4 constant region.
 49. The nucleic acidmolecule according to claim 45, wherein the antibody molecule is a humanantibody.
 50. A vector or recombinant cell, comprising the nucleicmolecule sequence of claim
 45. 51. A method of producing an antibodymolecule that specifically binds to the exosite 1 region of thrombin,comprising culturing the recombinant cell according to claim 50 underconditions such that the antibody molecule is expressed and recovered.52. An isolated or recombinant nucleic acid molecule encoding amonoclonal antibody molecule, comprising a variable light chain region(VL) comprising the amino acid sequence of SEQ ID NO: 14, a variableheavy chain region (VH) comprising the amino acid sequence of SEQ ID NO:2, and a human IgG constant region.
 53. The nucleic acid moleculeaccording to claim 52, wherein the human IgG constant region is a humanIgG1 constant region.
 54. The nucleic acid molecule according to claim52, wherein the human IgG constant region is a human IgG4 constantregion.
 55. A vector or recombinant cell, comprising the nucleic acidmolecule of claim
 52. 56. A vector or recombinant cell, comprising thenucleic acid molecule of claim
 53. 57. A vector or recombinant cell,comprising the nucleic acid molecule of claim
 54. 58. A method ofproducing an antibody molecule that specifically binds to the exosite 1region of thrombin, comprising culturing the recombinant cell accordingto claim 55 under conditions such that the antibody molecule isexpressed and recovered.